Gaines, Patrick J. (Fort Collins, CO, US)
Stinchcomb, Dan T. (Fort Collins, CO, US)
Wisnewski, Nancy (Fort Collins, CO, US)
What is claimed is:
1. An isolated nucleic acid molecule that hybridizes to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:1859, SEQ ID NO:1860, SEQ ID NO:1861, SEQ ID NO:1863, SEQ ID NO:1864, SEQ ID NO:1866, SEQ ID NO:1867, SEQ ID NO:1869, SEQ ID NO:1870, SEQ ID NO:1871, SEQ ID NO:1872, SEQ ID NO:1874, SEQ ID NO:1875, SEQ ID NO:1876, SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1880, SEQ ID NO:1881, SEQ ID NO:1882, SEQ ID NO:1884, SEQ ID NO:1885, SEQ ID NO:1886, SEQ ID NO:1887, SEQ ID NO:1889, SEQ ID NO:1890, SEQ ID NO:1891, SEQ ID NO:1892, SEQ ID NO:1893, SEQ ID NO:1894, SEQ ID NO:1895, SEQ ID NO:1896, SEQ ID NO:1898, SEQ ID NO:1899, SEQ ID NO:1900, SEQ ID NO:1901, SEQ ID NO:1903, SEQ ID NO:1904, SEQ ID NO:1905, SEQ ID NO:1906, SEQ ID NO:1907, SEQ ID NO:1908, SEQ ID NO:1909, SEQ ID NO:1910, SEQ ID NO:1911, SEQ ID NO:1912, SEQ ID NO:1913, SEQ ID NO:1914, SEQ ID NO:1916, SEQ ID NO:1917, SEQ ID NO:1918, SEQ ID NO:1919, SEQ ID NO:1921, SEQ ID NO:1922, SEQ ID NO:1923, SEQ ID NO:1924, SEQ ID NO:1926, SEQ ID NO:1927, SEQ ID NO:1928, SEQ ID NO:1929, and SEQ ID NO:1931, under conditions comprising (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C.
2. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule comprises a nucleic acid sequence that is at least about 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:1859, SEQ ID NO:1860, SEQ ID NO:1861, SEQ ID NO:1863, SEQ ID NO:1864, SEQ ID NO:1866, SEQ ID NO:1867, SEQ ID NO:1869, SEQ ID NO:1870, SEQ ID NO:1871, SEQ ID NO:1872,SEQ ID NO:1874,SEQ ID NO:1875,SEQ ID NO:1876,SEQ ID NO:1877,SEQ ID NO:1878,SEQ ID NO:1880,SEQ ID NO:1881,SEQ ID NO:1882, SEQ ID NO:1884, SEQ ID NO:1885, SEQ ID NO:1886, SEQ ID NO:1887, SEQ ID NO:1889, SEQ ID NO:1890, SEQ ID NO:1891, SEQ ID NO:1892, SEQ ID NO:1893, SEQ ID NO:1894, SEQ ID NO:1895, SEQ ID NO:1896, SEQ ID NO:1898, SEQ ID NO:1899, SEQ ID NO:1900, SEQ ID NO:1901, SEQ ID NO:1903, SEQ ID NO:1904, SEQ ID NO:1905, SEQ ID NO:1906, SEQ ID NO:1907, SEQ ID NO:1908, SEQ ID NO:1909, SEQ ID NO:1910, SEQ ID NO:1911, SEQ ID NO:1912, SEQ ID NO:1913, SEQ ID NO:1914,SEQ ID NO:1916,SEQ ID NO:1917,SEQ ID NO:1918,SEQ ID NO:1919, SEQ ID NO:1921, SEQ ID NO:1922, SEQ ID NO:1923, SEQ ID NO:1924, SEQ ID NO:1926, SEQ ID NO:1927, SEQ ID NO:1928, SEQ ID NO:1929, and SEQ ID NO:1931.
3. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule is selected from the group consisting of: a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:158,SEQ ID NO:159,SEQ ID NO:161,SEQ ID NO:162,SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:1859, SEQ ID NO:1860, SEQ ID NO:1861, SEQ ID NO:1863, SEQ ID NO:1864, SEQ ID NO:1866, SEQ ID NO:1867, SEQ ID NO:1869, SEQ ID NO:1870, SEQ ID NO:1871, SEQ ID NO:1872, SEQ ID NO:1874, SEQ ID NO:1875, SEQ ID NO:1876, SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1880, SEQ ID NO:1881, SEQ ID NO:1882, SEQ ID NO:1884, SEQ ID NO:1885, SEQ ID NO:1886, SEQ ID NO:1887, SEQ ID NO:1889, SEQ ID NO:1890, SEQ ID NO:1891, SEQ ID NO:1892, SEQ ID NO:1893, SEQ ID NO:1894, SEQ ID NO:1895, SEQ ID NO:1896, SEQ ID NO:1898, SEQ ID NO:1899, SEQ ID NO:1900, SEQ ID NO:1901, SEQ ID NO:1903, SEQ ID NO:1904, SEQ ID NO:1905, SEQ ID NO:1906, SEQ ID NO:1907, SEQ ID NO:1908, SEQ ID NO:1909,SEQ ID NO:1910,SEQ ID NO:1911,SEQ ID NO:1912,SEQ ID NO:1913, SEQ ID NO:1914, SEQ ID NO:1916, SEQ ID NO:1917, SEQ ID NO:1918, SEQ ID NO:1919, SEQ ID NO:1921, SEQ ID NO:1922, SEQ ID NO:1923, SEQ ID NO:1924, SEQ ID NO:1926, SEQ ID NO:1927, SEQ ID NO:1928, SEQ ID NO:1929, and SEQ ID NO:1931; and a nucleic acid molecule comprising an allelic variant of a nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:1859, SEQ ID NO:1860, SEQ ID NO:1861, SEQ ID NO:1863, SEQ ID NO:1864, SEQ ID NO:1866, SEQ ID NO:1867, SEQ ID NO:1869, SEQ ID NO:1870, SEQ ID NO:1871, SEQ ID NO:1872, SEQ ID NO:1874, SEQ ID NO:1875, SEQ ID NO:1876, SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1880, SEQ ID NO:1881, SEQ ID NO:1882, SEQ ID NO:1884, SEQ ID NO:1885, SEQ ID NO:1886, SEQ ID NO:1887, SEQ ID NO:1889, SEQ ID NO:1890, SEQ ID NO:1891, SEQ ID NO:1892, SEQ ID NO:1893, SEQ ID NO:1894, SEQ ID NO:1895, SEQ ID NO:1896, SEQ ID NO:1898, SEQ ID NO:1899, SEQ ID NO:1900, SEQ ID NO:1901, SEQ ID NO:1903, SEQ ID NO:1904, SEQ ID NO:1905, SEQ ID NO:1906, SEQ ID NO:1907, SEQ ID NO:1908, SEQ ID NO:1909, SEQ ID NO:1910, SEQ ID NO:1911, SEQ ID NO:1912, SEQ ID NO:1913, SEQ ID NO:1914, SEQ ID NO:1916, SEQ ID NO:1917, SEQ ID NO:1918, SEQ ID NO:1919, SEQ ID NO:1921, SEQ ID NO:1922, SEQ ID NO:1923, SEQ ID NO:1924, SEQ ID NO:1926, SEQ ID NO:1927, SEQ ID NO:1928, SEQ ID NO:1929, and SEQ ID NO:1931.
4. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule encodes a protein comprising an amino acid sequence that is at least about 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:154, SEQ ID NO:160, SEQ ID NO:163, SEQ ID NO:169, SEQ ID NO:1862, SEQ ID NO:1868, SEQ ID NO:1873, SEQ ID NO:1879, SEQ ID NO:1883, SEQ ID NO:1888, SEQ ID NO:1897, SEQ ID NO:1902, SEQ ID NO:1915, SEQ ID NO:1920, SEQ ID NO:1925, and SEQ ID NO:1930.
5. The nucleic acid molecule of claim 1, wherein said nucleic acid molecule encodes a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:154, SEQ ID NO:160, SEQ ID NO:163, SEQ ID NO:169, SEQ ID NO:1862, SEQ ID NO:1868, SEQ ID NO:1873, SEQ ID NO:1879, SEQ ID NO:1883, SEQ ID NO:1888, SEQ ID NO:1897, SEQ ID NO:1902, SEQ ID NO:1915, SEQ ID NO:1920, SEQ ID NO:1925, and SEQ ID NO:1930.
6. A recombinant molecule comprising a nucleic acid molecule as set forth in claim 1 operatively linked to a transcription control sequence.
7. A recombinant virus comprising a nucleic acid molecule as set forth in claim 1.
8. A recombinant cell comprising a nucleic acid molecule as set forth in claim 1.
9. A method to produce a protein encoded by a nucleic acid molecule that hybridizes to a nucleic acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:161, SEQ ID NO:164, SEQ ID NO:167, SEQ ID NO:170, SEQ ID NO:1860, SEQ ID NO:1863, SEQ ID NO:1866, SEQ ID NO:1869, SEQ ID NO:1871, SEQ ID NO:1874, SEQ ID NO:1876, SEQ ID NO:1880, SEQ ID NO:1884, SEQ ID NO:1886, SEQ ID NO:1889, SEQ ID NO:1891, SEQ ID NO:1893, SEQ ID NO:1895, SEQ ID NO:1898, SEQ ID NO:1900, SEQ ID NO:1903, SEQ ID NO:1905, SEQ ID NO:1907, SEQ ID NO:1909, SEQ ID NO:1911, SEQ ID NO:1913, SEQ ID NO:1916, SEQ ID NO:1918, SEQ ID NO:1921, SEQ ID NO:1923, SEQ ID NO:1926, SEQ ID NO:1928, and SEQ ID NO:1931 , under conditions comprising (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., said method comprising culturing a cell transformed with a nucleic acid molecule encoding said protein.
10. The method of claim 9, wherein said nucleic acid molecule encodes a protein having an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:8,SEQ ID NO:14,SEQ ID NO:20,SEQ ID NO:26,SEQ ID NO:32,SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:154, SEQ ID NO:160, SEQ ID NO:163, SEQ ID NO:169, SEQ ID NO:1862, SEQ ID NO:1868, SEQ ID NO:1873, SEQ ID NO:1879, SEQ ID NO:1883, SEQ ID NO:1888, SEQ ID NO:1897, SEQ ID NO:1902, SEQ ID NO:1915, SEQ ID NO:1920, SEQ ID NO:1925, and SEQ ID NO:1930.
11. The method of claim 9, wherein said nucleic acid molecule is selected from the group consisting of: a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:153, SEQ ID NO:156, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:165, SEQ ID NO:168, SEQ ID NO:1859, SEQ ID NO:1861, SEQ ID NO:1864, SEQ ID NO:1867, SEQ ID NO:1870, SEQ ID NO:1872, SEQ ID NO:1875, SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1881, SEQ ID NO:1882, SEQ ID NO:1885, SEQ ID NO:1887, SEQ ID NO:1890, SEQ ID NO:1892, SEQ ID NO:1894, SEQ ID NO:1896, SEQ ID NO:1899, SEQ ID NO:1901, SEQ ID NO:1904, SEQ ID NO:1906,SEQ ID NO:1908,SEQ ID NO:1910,SEQ ID NO:1912,SEQ ID NO:1914, SEQ ID NO:1917, SEQ ID NO:1919, SEQ ID NO:1922, SEQ ID NO:1924, SEQ ID NO:1927, and SEQ ID NO:1929; and a nucleic acid molecule comprising an allelic variant of a nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:153, SEQ ID NO:156, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:165, SEQ ID NO:168, SEQ ID NO:1859, SEQ ID NO:1861, SEQ ID NO:1864, SEQBD NO:1867, SEQ ID NO:1870, SEQ ID NO:1872, SEQ ID NO:1875, SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1881, SEQ ID NO:1882, SEQ ID NO:1885, SEQ ID NO:1887, SEQ ID NO:1890, SEQ ID NO:1892, SEQ ID NO:1894, SEQ ID NO:1896, SEQ ID NO:1899, SEQ ID NO:1901, SEQ ID NO:1904, SEQ ID NO:1906, SEQ ID NO:1908, SEQ ID NO:1910, SEQ ID NO:1912, SEQ ID NO:1914,SEQ ID NO:1917,SEQ ID NO:1919,SEQ ID NO:1922,SEQ ID NO:1924, SEQ ID NO:1927, and SEQ ID NO:1929.
12. An isolated protein selected from the group consisting of: (a) an isolated protein encoded by a nucleic acid molecule that hybridizes to a nucleic acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:161, SEQ ID NO:164, SEQ ID NO:167, SEQ ID NO:170, SEQ ID NO:1860, SEQ ID NO:1863, SEQ ID NO:1866, SEQ ID NO:1869, SEQ ID NO:1871, SEQ ID NO:1874, SEQ ID NO:1876, SEQ ID NO:1880, SEQ ID NO:1884, SEQ ID NO:1886, SEQ ID NO:1889, SEQ ID NO:1891, SEQ ID NO:1893, SEQ ID NO:1895, SEQ ID NO:1898, SEQ ID NO:1900, SEQ ID NO:1903, SEQ ID NO:1905, SEQ ID NO:1907, SEQ ID NO:1909, SEQ ID NO:1911, SEQ ID NO:1913, SEQ ID NO:1916, SEQ ID NO:1918, SEQ ID NO:1921, SEQ ID NO:1923, SEQ ID NO:1926, SEQ ID NO:1928, and SEQ ID NO:1931 , under conditions comprising (1) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (2) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C.; and (b) an isolated protein comprising an amino acid sequence that is at least about 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:154, SEQ ID NO:160, SEQ ID NO:163, SEQ ID NO:169,SEQ ID NO:1862,SEQ ID NO:1868,SEQ ID NO:1873,SEQ ID NO: 1879, SEQ ID NO:1883, SEQ ID NO:1888, SEQ ID NO:1897, SEQ ID NO:1902, SEQ ID NO:1915, SEQ ID NO:1920, SEQ ID NO:1925, and SEQ ID NO:1930.
13. The protein of claim 12, wherein said nucleic acid molecule comprises a nucleic acid sequence that is at least about 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:153, SEQ ID NO:156, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:165, SEQ ID NO:168, SEQ ID NO:1859, SEQ ID NO:1861, SEQ ID NO:1864, SEQ ID NO:1867, SEQ ID NO:1870, SEQ ID NO:1872, SEQ ID NO:1875, SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1881, SEQ ID NO:1882, SEQ ID NO:1885, SEQ ID NO:1887, SEQ ID NO:1890, SEQ ID NO:1892, SEQ ID NO:1894, SEQ ID NO:1896, SEQ ID NO:1899, SEQ ID NO:1901, SEQ ID NO:1904, SEQ ID NO:1906, SEQ ID NO:1908, SEQ ID NO:1910, SEQ ID NO:1912, SEQ ID NO:1914, SEQ ID NO:1917, SEQ ID NO:1919, SEQ ID NO:1922, SEQ ID NO:1924, SEQ ID NO:1927, and SEQ ID NO:1929.
14. The protein of claim 12, wherein said nucleic acid molecule is selected from the group consisting of: a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13,SEQ ID NO:16,SEQ ID NO:19,SEQ ID NO:22,SEQ ID NO:25,SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:153, SEQ ID NO:156, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:165, SEQ ID NO:168, SEQ ID NO:1859, SEQ ID NO:1861, SEQ ID NO:1864, SEQ ID NO:1867, SEQ ID NO:1870, SEQ ID NO:1872, SEQ ID NO:1875, SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1881, SEQ ID NO:1882, SEQ ID NO:1885, SEQ ID NO:1887, SEQ ID NO:1890, SEQ ID NO:1892, SEQ ID NO:1894, SEQ ID NO:1896, SEQ ID NO:1899, SEQ ID NO:1901, SEQ ID NO:1904, SEQ ID NO:1906, SEQ ID NO:1908, SEQ ID NO:1910, SEQ ID NO:1912, SEQ ID NO:1914, SEQ ID NO:1917, SEQ ID NO:1919, SEQ ID NO:1922, SEQ ID NO:1924, SEQ ID NO:1927, and SEQ ID NO:1929; and a nucleic acid molecule comprising an allelic variant of a nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:153, SEQ ID NO:156, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:165, SEQ ID NO:168, SEQ ID NO:1859, SEQ ID NO:1861, SEQ ID NO:1864, SEQ ID NO:1867, SEQ ID NO:1870, SEQ ID NO:1872, SEQ ID NO:1875, SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1881, SEQ ID NO:1882, SEQ ID NO:1885, SEQ ID NO:1887, SEQ ID NO:1890, SEQ ID NO:1892, SEQ ID NO:1894, SEQ ID NO:1896, SEQ ID NO:1899, SEQ ID NO:1901, SEQ ID NO:1904, SEQ ID NO:1906, SEQ ID NO:1908, SEQ ID NO:1910, SEQ ID NO:1912, SEQ ID NO:1914, SEQ ID NO:1917, SEQ ID NO:1919, SEQ ID NO:1922, SEQ ID NO:1924, SEQ ID NO:1927, and SEQ ID NO:1929.
15. The protein of claim 12, wherein said protein comprises an amino acid sequence that is at least about 75% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:154, SEQ ID NO:160, SEQ ID NO:163, SEQ ID NO:169, SEQ ID NO:1862, SEQ ID NO:1868, SEQ D NO:1873, SEQ ID NO:1879, SEQ ID NO:1883, SEQ ID NO:1888, SEQ ID NO:1897, SEQ ID NO:1902, SEQ ID NO:1915, SEQ ID NO:1920, SEQ ID NO:1925, and SEQ ID NO:1930.
16. The protein of claim 12, wherein said protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:154, SEQ ID NO:160, SEQ ID NO:163, SEQ ID NO:169, SEQ ID NO:1862, SEQ ID NO:1868, SEQ ID NO:1873, SEQ ID NO:1879, SEQ ID NO:1883, SEQ ID NO:1888, SEQ ID NO:1897, SEQ ID NO:1902, SEQ ID NO:1915, SEQ ID NO:1920, SEQ ID NO:1925, and SEQ ID NO:1930.
17. An isolated antibody that selectively binds to a protein as set forth in claim 12.
18. A method to identify a compound capable of inhibiting activity of an isolated protein of claim 12, said method comprising contacting an isolated protein of claim 12 with a putative inhibitory compound under conditions in which, in the absence of said compound, said protein has activity; and determining if said putative inhibitory compound inhibits said activity.
19. A kit to identify a compound capable of inhibiting activity of an isolated protein of claim 12, said test kit comprising an isolated protein of claim 12 and a means for determining the extent of inhibition of said activity in the presence of a putative inhibitory compound.
20. A composition comprising an excipient and a compound selected from the group consisting of: (a) an isolated nucleic acid molecule that hybridizes to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ D NO:30,SEQ ID NO:31,SEQ ID NO:33,SEQ ID NO:34,SEQ ID NO:36,SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:1859, SEQ ID NO:1860, SEQ ID NO:1861, SEQ ID NO:1863, SEQ ID NO:1864, SEQ ID NO:1866, SEQ ID NO:1867, SEQ ID NO:1869, SEQ ID NO:1870, SEQ ID NO:1871, SEQ ID NO:1872, SEQ ID NO:1874, SEQ ID NO:1875, SEQ ID NO:1876, SEQ ID NO:1877,SEQ ID NO:1878,SEQBD NO:1880,SEQ ID NO:1881,SEQ ID NO:1882, SEQ ID NO:1884, SEQ ID NO:1885, SEQ ID NO:1886, SEQ ID NO:1887, SEQ ID NO:1889, SEQ ID NO:1890, SEQ ID NO:1891, SEQ ID NO:1892, SEQ ID NO:1893, SEQ ID NO:1894, SEQ ID NO:1895, SEQ ID NO:1896, SEQ ID NO:1898, SEQ ID NO:1899, SEQ ID NO:1900, SEQ ID NO:1901, SEQ ID NO:1903, SEQ ID NO:1904, SEQ ID NO:1905, SEQ ID NO:1906, SEQ ID NO:1907, SEQ ID NO:1908, SEQ ID NO:1909, SEQ ID NO:1910, SEQ ID NO:1911, SEQ ID NO:1912, SEQ ID NO:1913, SEQ ID NO:1914,SEQ ID NO:1916,SEQ ID NO:1917,SEQ ID NO:1918,SEQ ID NO:1919, SEQ ID NO:1921, SEQ ID NO:1922, SEQ ID NO:1923, SEQ ID NO:1924, SEQ ID NO:1926, SEQ ID NO:1927, SEQ ID NO:1928, SEQ ID NO:1929, and SEQ ID NO:1931, under conditions comprising (1) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (2) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C.; (b) an isolated protein encoded by a nucleic acid molecule that hybridizes to a nucleic acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:161, SEQ ID NO:164, SEQ ID NO:167, SEQ ID NO:170, SEQ ID NO:1860, SEQ ID NO:1863, SEQ ID NO:1866, SEQ ID NO:1869, SEQ ID NO:1871, SEQ ID NO:1874, SEQ ID NO:1876, SEQ ID NO:1880, SEQ ID NO:1884, SEQ ID NO:1886, SEQ ID NO:1889, SEQ ID NO:1891, SEQ ID NO:1893, SEQ ID NO:1895, SEQ ID NO:1898, SEQ ID NO:1900, SEQ ID NO:1903, SEQ ID NO:1905, SEQ ID NO:1907, SEQ ID NO:1909, SEQ ID NO:1911, SEQ ID NO:1913,SEQ ID NO:1916,SEQ ID NO:1918,SEQ ID NO:1921,SEQ ID NO:1923, SEQ ID NO:1926, SEQ ID NO:1928, and SEQ ID NO:1931, under conditions comprising (1) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (2) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C.; and (c) an isolated antibody that selectively binds to a protein encoded by a nucleic acid molecule that hybridizes to a nucleic acid sequence selected from the group consisting of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:161, SEQ ID NO:164, SEQ ID NO:167, SEQ ID NO:170, SEQ ID NO:1860, SEQ ID NO:1863, SEQ ID NO:1866, SEQ ID NO:1869, SEQ ID NO:1871, SEQ ID NO:1874, SEQ ID NO:1876, SEQ ID NO:1880, SEQ ID NO:1884, SEQ ID NO:1886, SEQ ID NO:1889, SEQ ID NO:1891, SEQ ID NO:1893, SEQ ID NO:1895, SEQ ID NO:1898, SEQ ID NO:1900, SEQ ID NO:1903, SEQ ID NO:1905, SEQ ID NO:1907, SEQ ID NO:1909, SEQ ID NO:1911, SEQ ID NO:1913,SEQ ID NO:1916,SEQ ID NO:1918,SEQ ID NO:1921,SEQ ID NO:1923, SEQ ID NO:1926, SEQ ID NO:1928, and SEQ ID NO:1931, under conditions comprising (1) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (2) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C.
21. The composition of claim 20, wherein said composition further comprises a component selected from the group consisting of an adjuvant and a carrier.
22. A method to protect an animal, said method comprising administering to said animal a composition of claim 20.
23. An isolated nucleic acid molecule expressed by a tissue selected from the group consisting of a flea HMT tissue and a flea HNC tissue, identified by a method comprising: (a) constructing a cDNA library enriched for HMT or HNC expressed sequences; and (b) identifying a nucleic acid molecule in said library.
24. The nucleic acid molecule of claim 23, wherein said nucleic acid molecule encodes a protein selected from the group consisting of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ED NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:154, SEQ ID NO:160, SEQ ID NO:163, SEQ ID NO:169, SEQ ID NO:1862, SEQ ID NO:1868, SEQ ID NO:1873, SEQ ID NO:1879, SEQ ID NO:1883, SEQ ID NO:1888, SEQ ID NO:1897, SEQ ID NO:1902, SEQ ID NO:1915, SEQ ID NO:1920, SEQ ID NO:1925, SEQ ID NO:1930, and a protein encoded by a nucleic acid sequence selected from the group consisting of a nucleic acid sequence of Table I, a nucleic acid sequence of Table II, a nucleic acid sequence of Table III, and a nucleic acid sequence of Table IV.
25. An isolated antibody that selectively binds to a protein as set forth in claim 24.
26. The nucleic acid molecule of claim 23, wherein said nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of a nucleic acid sequence of Table I, a nucleic acid sequence of Table II, a nucleic acid sequence of Table II, and a nucleic acid sequence of Table IV.
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 60/128,704, entitled “NOVEL FLEA HEAD, NERVE CORD, HINDGUT AND MALPIGHIAN TUBULE NUCLEIC ACID MOLECULES, PROTEINS AND USES THEREOF”, filed Apr. 9, 1999.
FIELD OF THE INVENTION
[0002] The present invention relates to nucleic acid molecules isolated from the head and nerve cord of a flea, nucleic acid molecules isolated from the hindgut and Malpighian tubule of a flea, proteins encoded by such nucleic acid molecules, antibodies raised against such proteins, and inhibitors of such proteins. The present invention also includes therapeutic compositions comprising such nucleic acid molecules, proteins, antibodies, and/or other inhibitors, as well as uses thereof.
BACKGROUND OF THE INVENTION
[0003] Flea infestation of animals is a health and economic concern because fleas are known to cause and/or transmit a variety of diseases. Fleas directly cause a variety of diseases, including allergies, and also carry a variety of infectious agents including, but not limited to, endoparasites (e.g., nematodes, cestodes, trematodes and protozoa), bacteria and viruses. In particular, the bites of fleas are a problem for animals maintained as pets because the infestation becomes a source of annoyance not only for the pet but also for the pet owner who may find his or her home generally contaminated with insects. As such, fleas are a problem not only when they are on an animal but also when they are in the general environment of the animal.
[0004] Bites from fleas are a particular problem because they not only can lead to disease transmission but also can cause a hypersensitive response in animals which is manifested as disease. For example, bites from fleas can cause an allergic disease called flea allergic (or allergy) dermatitis (FAD). A hypersensitive response in animals typically results in localized tissue inflammation and damage, causing substantial discomfort to the animal.
[0005] The medical importance of flea infestation has prompted the development of reagents capable of controlling flea infestation. Commonly encountered methods to control flea infestation are generally focused on use of insecticides. While some of these products are efficacious, most, at best, offer protection of a very limited duration. Furthermore, many of the methods are often not successful in reducing flea populations. In particular, insecticides have been used to prevent flea infestation of animals by adding such insecticides to shampoos, powders, collars, sprays, spot-on formulations foggers and liquid bath treatments (i.e., dips). Reduction of flea infestation on the pet has been unsuccessful for one or more of the following reasons: failure of owner compliance (frequent administration is required); behavioral or physiological intolerance of the pet to the pesticide product or means of administration; and the emergence of flea populations resistant to the prescribed dose of pesticide.
[0006] Thus, there remains a need to develop a reagent and a method to protect animals from flea infestation.
SUMMARY OF THE INVENTION
[0007] The present invention relates to a novel product and process for protection of animals from flea infestation.
[0008] The present invention provides flea head and nerve cord (HNC) proteins and flea hindgut and Malpighian tubule (HMT) proteins; nucleic acid molecules encoding flea HNC proteins and flea HMT proteins; antibodies raised against such proteins (i.e., anti-flea HNC antibodies and anti-flea HMT antibodies respectively); mimetopes of such proteins or antibodies; and compounds that inhibit flea HNC or HMT activity (i.e, inhibitory compounds or inhibitors).
[0009] The present invention also includes methods to obtain such proteins, mimetopes, nucleic acid molecules, antibodies and inhibitory compounds. The present invention also includes the use of proteins and antibodies to identify such inhibitory compounds as well as assay kits to identify such inhibitory compounds. Also included in the present invention are therapeutic compositions comprising proteins, mimetopes, nucleic acid molecules, antibodies and inhibitory compounds of the present invention including protective compounds derived from a protein of the present invention that inhibit the activity of HNC and/or HMT proteins; also included are uses of such therapeutic compounds to reduce flea infestation.
[0010] One embodiment of the present invention is an isolated nucleic acid molecule that hybridizes with a nucleic acid sequence having SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:161, SEQID NO:162, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:1859, SEQ ID NO:1860, SEQ ID NO:1861, SEQ ID NO:1863, SEQ ID NO:1864, SEQ ID NO:1866, SEQ ID NO:1867, SEQ ID NO:1869, SEQ ID NO:1870, SEQ ID NO:1871, SEQ ID NO:1872, SEQ ID NO:1874, SEQ ID NO:1875, SEQ ID NO:1876, SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1880, SEQ ID NO:1881, SEQ ID NO:1882, SEQ ID NO:1884, SEQ ID NO:1885, SEQ ID NO:1886, SEQ ID NO:1887, SEQ ID NO:1889, SEQ ID NO:1890, SEQ ID NO:1891, SEQ ID NO:1892, SEQ ID NO:1893, SEQ ID NO:1894, SEQ ID NO:1895, SEQ ID NO:1896, SEQ ID NO:1898, SEQ ID NO:1899, SEQ ID NO:1900, SEQ ID NO:1901, SEQ ID NO:1903, SEQ ID NO:1904, SEQ ID NO:1905, SEQ ID NO:1906, SEQ ID NO:1907, SEQIID NO:1908, SEQ ID NO:1909, SEQ ID NO:1910, SEQ ID NO:1911, SEQID NO:1912, SEQ ID NO:1913, SEQ ID NO:1914, SEQ ID NO:1916, SEQ ID NO:1917, SEQ ID NO:1918, SEQ ID NO:1919, SEQ ID NO:1921, SEQ ID NO:1922, SEQ ID NO:1923, SEQ ID NO:1924, SEQ ID NO:1926, SEQ ID NO:1927, SEQ ID NO:1928, SEQ ID NO:1929, and/or SEQ ID NO:1931 under conditions that allow less than or equal to about 30% base pair mismatch. Another embodiment of the present invention is an isolated nucleic acid molecule that hybridizes with a nucleic acid molecule selected from the group consisting of a nucleic acid sequence of Table I, Table II, Table m and/or Table IV, or a nucleic acid sequence complementary to a nucleic acid sequence of Table I, Table II, Table III and/or Table IV under conditions that allow less than or equal to about 30% base pair mismatch.
[0011] Another embodiment of the present invention is an isolated nucleic acid molecule having nucleic acid sequence that is at least about 70% identical to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:1859, SEQ ID NO:1860, SEQ ID NO:1861, SEQ ID NO:1863, SEQ ID NO:1864, SEQ ID NO:1866, SEQ ID NO:1867, SEQ ID NO:1869, SEQ ID NO:1870, SEQ ID NO:1871, SEQ ID NO:1872, SEQ ID NO:1874, SEQ ID NO:1875, SEQ ID NO:1876, SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1880, SEQ ID NO:1881, SEQ ID NO:1882, SEQ ID NO:1884, SEQ ID NO:1885, SEQ ID NO:1886, SEQ ID NO:1887, SEQ ID NO:1889, SEQ ID NO:1890, SEQ ID NO:1891, SEQ ID NO:1892, SEQ ID NO:1893, SEQ ID NO:1894, SEQ ID NO:1895, SEQ ID NO:1896, SEQ ID NO:1898, SEQ ID NO:1899, SEQ ID NO:1900, SEQ ID NO:1901, SEQ ID NO:1903, SEQ ID NO:1904, SEQ ID NO:1905, SEQ ID NO:1906, SEQ ID NO:1907,SEQ ID NO:1908,SEQ ID NO:1909,SEQ ID NO:1910,SEQ ID NO:1911, SEQ ID NO:1912, SEQ ID NO:1913, SEQ ID NO:1914, SEQ ID NO:1916, SEQ ID NO:1917,SEQ ID NO:1918,SEQ ID NO:1919,SEQ ID NO:1921,SEQ ID NO:1922, SEQ ID NO:1923, SEQ ID NO:1924, SEQ ID NO:1926, SEQ ID NO:1927, SEQ ID NO:1928, SEQ ID NO:1929, and/or SEQ ID NO:1931 and/or a nucleic acid sequence of Table I, Table II, Table III and/or Table IV or complements thereof.
[0012] The present invention also relates to recombinant molecules, recombinant viruses and recombinant cells that include a nucleic acid molecule of the present invention. Also included are methods to produce such nucleic acid molecules, recombinant molecules, recombinant viruses and recombinant cells.
[0013] Another embodiment of the present invention includes an isolated flea HMT and/or HNC protein that is at least about 70% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:154, SEQ ID NO:160, SEQ ID NO:163, SEQ ID NO:169, SEQ ID NO:1862, SEQ ID NO:1868, SEQ ID NO:1873, SEQ ID NO:1879, SEQ ID NO:1883, SEQ ID NO:1888, SEQ ID NO:1897, SEQ ID NO:1902, SEQ ID NO:1915, SEQ ID NO:1920, SEQ ID NO:1925, and/or SEQ ID NO:1930, and/or an amino acid sequence encoded by a nucleic acid sequence of Table I, Table II, Table III and/or Table IV, and fragments thereof, wherein such fragments can elicit an immune response against respective flea proteins or have activity comparable to respective flea proteins.
[0014] Another embodiment of the present invention includes an isolated protein encoded by a nucleic acid molecule that hybridizes with the complement of a nucleic acid sequence having SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:153, SEQ ID NO:156, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:165, SEQ ID NO:168, SEQ ID NO:1859, SEQ ID NO:1861, SEQ ID NO:1864, SEQ ID NO:1867, SEQ ID NO:1870, SEQ ID NO:1872, SEQ ID NO:1875, SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1881, SEQ ID NO:1882, SEQ ID NO:1885, SEQ ID NO:1887, SEQ ID NO:1890, SEQ ID NO:1892, SEQ ID NO:1894, SEQ ID NO:1896, SEQ ID NO:1899, SEQ ID NO:1901, SEQ ID NO:1904, SEQ ID NO:1906, SEQ ID NO:1908, SEQ ID NO:1910, SEQ ID NO:1912, SEQ ID NO:1914, SEQ ID NO:1917, SEQ ID NO:1919, SEQ ID NO:1922, SEQ ID NO:1924, SEQ ID NO:1927, and/or SEQ ID NO:1929 and/or a nucleic acid sequence of Table I, Table II, Table III and/or Table IV, under conditions that allow less than or equal to about 30% base pair mismatch.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The present invention provides for nucleic acid molecules isolated from the head and/or nerve cord of a flea, nucleic acid molecules isolated from the hindgut and/or Malpighian tubule of a flea, proteins encoded by such nucleic acid molecules, antibodies raised against such proteins, and inhibitors of such proteins. As used herein, nucleic acid molecules isolated from the head and/or nerve cord of a flea and proteins encoded by such nucleic acid molecules are also referred to as flea HNC, or HNC, nucleic acid molecules and proteins respectively; and nucleic molecules isolated from the hindgut and/or Malpighian tubules of a flea and proteins encoded by such nucleic acid molecules are referred to as flea HMT or HMT, nucleic acid molecules and proteins respectively. HNC nucleic acid molecules and HMT nucleic acid molecules of the present invention are nucleic acid molecules that are primarily expressed in flea HNC tissues and HMT tissues respectively, but which may be expressed in cells derived from flea tissues other than HNC and HMT. HNC and HMT nucleic acid molecules and proteins of the present invention can be isolated from a flea or prepared recombinantly or synthetically. HMT and HNC nucleic acid molecules of the present invention can be RNA or DNA; examples of nucleic acid molecules include, but are not limited to, complementary DNA (cDNA) molecules, genomic DNA molecules, synthetic DNA molecules, DNA molecules which are specific tags for messenger RNA derived from HMT and HNC tissues, and corresponding MRNA molecules. As used herein, the phrases “HMT and/or HNC protein” and “HMT and HNC protein” refer to a protein expressed by a flea HMT tissue, by a flea HNC tissue, or by both flea HMT and HNC tissues. As used herein, the phrases “HMT and/or HNC nucleic acid molecule” and “HMT and HNC nucleic acid molecule” refer to a nucleic acid molecule that can be isolated from a HMT cDNA library, from a HNC cDNA library, or from both libraries, or a gene corresponding thereto.
[0016] The present invention provides for nucleic acid molecules containing partial or full-length coding regions that encode one or more of the following flea proteins: an allantoinase (ALN) protein, a chitin-binding protein (CBP) protein, a sodium/potassium ATPase beta subunit (NKAB) protein, a ligand-gated chloride channel (LGIC) protein, an ANON/23DA (ANON) protein, a malvolio (MALV) protein, an odorant-binding protein-like (OS-D) protein, a N-methyl-D-aspartate receptor associated (NMDA) protein, a chemical sense related lipophilic ligand binding protein-like (CLBP) protein, a Sodium/Hydrogen Transporter-like (NAH) protein, a Chloride Intracellular Channel-like (CLIC) protein, aPeritrophin-like (PL2) protein, aPeritrophin-like (PL3) protein, aPeritrophin-like (PL4) protein, a synaptic vesicle 2B-like (SVP) protein, a voltage-gated Chloride-like (VGCC) protein, an anoxia upregulated protein-like (AUP) protein, and a neuroendocrine specific 7B2-like (7B2) protein. Such nucleic acid molecules are referred to as ALN nucleic acid molecules, CBP nucleic acid molecules, NKAB nucleic acid molecules, LGIC nucleic acid molecules, ANON nucleic acid molecules, MALV nucleic acid molecules, OS-D nucleic acid molecules, NMDA nucleic acid molecules, CLBP nucleic acid molecules, NAH nucleic acid molecules, CLIC nucleic acid molecules, PL2 nucleic acid molecules, PL3 nucleic acid molecules, PL4 nucleic acid molecules, SVP nucleic acid molecules, VGCC nucleic acid molecules, AUP nucleic acid molecules, and 7B2 nucleic acid molecules respectively and are described herein in detail below.
[0017] Allantoinase is involved in the catalysis of the reaction converting allantoin to allantoic acid. This is a middle step in purine catabolism, which in insects results in the secretion of urea as the end product. The enzyme is located in the peroxisomes of the liver and kidney in amphibians. There is no known mammalian homologue to allantoinase, as mammals secrete uric acid, a precursor to allantoin. As such, flea allantoinase represents a novel target for anti-flea vaccines and chemotherapeutic drugs.
[0018] The function of chitin binding protein is largely unknown. A chitinase-like protein of
[0019] Na+/K+ATPase is involved in the hydrolysis of ATP to power the transport of Na+ out of and K+ into cells. It is responsible for establishing the Na+ gradient across plasma membranes, which is then used by cells for a number of functions including sugar and amino acid transport, diuresis and nerve cell signaling. The Na+/K+ ATPase pump is a trimer of a 100-kilodalton (kDa) alpha (α) subunit, a 40-kDa beta (β) subunit, and a 6-kDa gamma (γ) subunit. Most insects express three isotypes of the β subunit, each being expressed in a tissue and cell-type dependent manner. The α subunit has 8 transmembrane domains whereas the β and γ subunits have just one. The α subunit mediates ATPase and ion transporting activities and together with the γ subunit comprises the site for cardiac glycoside (ouabain) binding. The β subunit is required for detectable pump activity, and is thought to have roles in stability, localization, and determining cation specificity. As such, a flea NKAB protein of the present invention represents a novel target for anti-flea vaccines and chemotherapeutic drugs.
[0020] Ligand-gated ion channel family proteins have been shown to transmit neural signals in response to binding neurotransmitters such as GABA, glycine, and glutamate. GABA and glycine receptors transmit inhibitory signals whereas glutamate receptors transmit excitatory signals. This family of proteins is the target for many drugs affecting neural signaling, and also for several families of insecticides including cyclodienes, pyrethroids, and phenyl pyrazoles. Northern blot analysis indicates that the mRNA corresponding to a LGIC nucleic acid molecule of the present invention is only expressed in HMT tissue, which suggests a role in the regulation or mediation of diuresis. Without being bound by theory, assuming protein expression correlates with the mRNA expression, flea LGIC may represent the first of this family of receptors shown to be exclusively expressed in renal tissue. Sequence analysis shows that a flea LGIC protein is distinct from other subfamilies of ligand-gated ion channels, and thus may represent a new subfamily. As such, a flea LGIC protein of the present invention represents a novel target for anti-flea vaccines and chemotherapeutic drugs.
[0021] The function of ANON/23DA protein largely unknown. The ANON/23DA gene is reported to be linked to the MAD gene in Drosophila, though it is not known if ANON/23DA and MAD are functionally related. ANON/23DA may also have functional similarity to human probable membrane receptor protein pHPS1-2, which is similar to rhodopsin/beta-adrenergic receptor which plays an important role in kidney function. As such, a flea ANON/23DA protein of the present invention represents a novel target for anti-flea vaccines and chemotherapeutic drugs.
[0022] Drosophila malvolio shows high sequence homology to mammalian natural resistance associated proteins (NRAMPs) and to yeast Smf1, which are proteins that transport divalent cations, specifically Mn++, Zn++, and Fe++. NRAMPs have also been shown be similar to ATPase transporters and use ATP as an energy source. There are two types of NRAMP proteins, NRAMP1 and NRAMP2. NRAMP1 is expressed exclusively on macrophages and is responsible for preventing intracellular replication of microbes. NRAMP2 is expressed in several cell and tissue types, including mouse intestinal epithelia. Flea malvolio proteins of the present invention appear to be most similar to NRAMP1. As such, a flea malvolio protein of the present invention represents a novel target for anti-flea vaccines and chemotherapeutic drugs.
[0023] The function of OS-D proteins is largely unknown. An OS-D nucleic acid molecule isolated from a
[0024] NMDA receptors are a subtype of glutamate-gated ion channels. All glutamate-gated ion channels transmit Na+ and K+ when stimulated, resulting in a depolarization of the membrane potential. NMDA receptors also transport Ca++ into cells upon stimulation, which distinguishes NMDA receptors from the other glutamate-gated ion channels. NMDA receptors play an important role in glutamate excitotoxicity, which has been linked to a number of neurodegenerative disorders such as focal cerebral ischemia (stroke), Parkinson's disease, Huntington's chorea, Alzheimer's disease, schizophrenia and epilepsy. It is thought that the Ca++ influx in open NMDA channels is the mediator for these diseases, since the increase in intracellular Ca++ concentration leads to the induction of metabolic changes in the cell, including the activation of Ca++ dependent proteases and production of free-oxygen radicals. As such, a flea NMDA protein of the present invention represents a novel target for anti-flea vaccines and chemotherapeutic drugs.
[0025] CLBP proteins of the present invention appear to fall into the family of PBP/GOBP proteins (pheromone binding protein/general odorant binding protein) based on sequence homology with members of this family (30% identity with PBPRP-2, pheromone binding protein related protein #2 of
[0026] Peritrophins, including flea PL2, PL3 and PL4 proteins of the present invention, are a family of putative chitin-binding proteins that comprise a structural component of the peritrophic matrix, an acellular membrane composed of proteins and sugars, most commonly chitin which forms a barrier between the contents of an ingested meal and the gut epithelia. Peritrophin-like proteins have also been shown to be present in the trachea of Drosophila embryos, indicating that such proteins may have additional roles outside the midgut. The function of the peritrophin-like proteins in adult fleas is not clear, since adult fleas do not produce a peritrophic matrix in the gut. Peritrophins have been investigated as targets for immunological control of hematophagous insects including the sheep blowfly,
[0027] In general, voltage-gated chloride channels (VGCC) maintain resting epithelial and neural membrane potentials and prevent hyperexcitability (sustained contraction) in muscle cells. In Drosophila Malpighian tubules, the diuretic hormone leukokinin has been shown to stimulate voltage-gated chloride channels in the stellate cells by increasing intracellular calcium levels. The flea VGCC protein sequence of the present invention contains an EF-hand calcium binding motif, indicating potential regulation by calcium ions, and thus a possible link to leukokinins and diuresis. Chloride channels are critical for diuresis since chloride is the primary anion driving diuresis and is required to help neutralize the sodium and potassium cations that are secreted into the lumen in response to diuretic peptide. The mRNA for the VGCC of the present invention has been shown to be HMT-specific in adult fleas, indicating a potential role in diuresis. As such, a flea VGCC of the present invention represents a novel target for anti-flea vaccines and chemotherapeutic drugs.
[0028] The CLIC family of chloride channels are voltage-gated chloride channels that are expressed on a variety of vesicles and are thought to act in concert with the V-ATPase pump to regulate the pH of the vesicle interior. Members of the CLIC family have also been shown to be expressed on the plasma membrane, again, in association with the V-ATPase pump. In humans, a homologous protein has been shown to be expressed on the plasma membrane in epithelial tissues, suggesting a possible role in transepithelial chloride transport and in cows, an antibody against a homologous channel has been shown to inhibit all chloride conductance in kidney microsomes. If the CLIC gene product is indeed involved in transepithelial chloride transport in HMT tissues, it likely plays a critical role in mediating diuresis. As such, a flea CLIC of the present invention represents a novel target for anti-flea vaccines and chemotherapeutic drugs.
[0029] The NAH exchanger uses the proton gradient in the lumen of the Malpighian tubule to power the transport of sodium ions across the apical membrane into the lumen. The transport of sodium ions across the Malpighian tubule epithelia is induced by diuretic peptide and is a critical step in the induction of diuresis. The Northern blot analysis described herein indicates that NAH mRNA is upregulated within 15 minutes of feeding in adults, which is consistent with a molecule having a role in diuresis. In many insects, sodium has been shown to be the principle ion driving diuresis. The NAH exchanger has been shown to be located on the apical membrane in the Malpighian tubules, but may also be located in the hindgut and rectum. If located in the hindgut and rectum, it could be accessible to antibody attack on either the basolateral or apical membranes. As such, a flea NAH of the present invention represents a novel target for anti-flea vaccines and chemotherapeutic drugs.
[0030] SVP proteins have structural and sequence conservation with a bacterial family of proton co-transporters, with the mammalian proton/glucose transporter, and with organic ion transporters. SVP has 12 putative transmembrane regions that arise from an internal duplication. In mammals, it is located on neural and endocrine vesicles and is thought to function in the uptake of neurotransmitters into vesicles utilizing the proton gradient. Neurotransmitters in turn regulate the activity the ion channels on these membranes. In the Malpighian tubules, the activity of the ion channels determines the rate of diuresis, or fluid secretion from the hemolymph into the lumen. Thus, inhibiting the transport of neurotransmitters in the HMT tissues may have significant effects on the functions of these tissues. As such, a flea SVP of the present invention represents a novel target for anti-flea vaccines and chemotherapeutic drugs.
[0031] The function of flea AUP proteins is largely unknown.
[0032] A flea 7B2 protein has some BLAST homology to the neuroendocrine protein 7B2 from various organisms, including Drosophila,
[0033] Flea allantoinase nucleic acid molecules of known length isolated from
[0034] The present invention also provides for HMT and HNC DNA molecules that are specific tags for messenger RNA molecules derived from HMT and HNC tissues. Such DNA molecules can correspond to an entire or partial sequence of a messenger RNA, and therefore, a DNA molecule corresponding to such a messenger RNA molecule (i.e. a cDNA molecule), can encode a full-length or partial-length protein. A nucleic acid molecule encoding a partial-length protein can be used directly as a probe or indirectly to generate primers to identify and/or isolate a cDNA nucleic acid molecule encoding a corresponding, or structurally related, full-length protein. Such a partial cDNA nucleic acid molecule can also be used in a similar manner to identify a genomic nucleic acid molecule, such as a nucleic acid molecule that contains the complete gene including regulatory regions, exons and introns. Methods for using partial HMT and HNC cDNA molecules and sequences to isolate full-length transcripts and corresponding cDNA molecules are described in the examples herein below.
[0035] The proteins and nucleic acid molecules of the present invention can be obtained from their natural source, or can be produced using, for example, recombinant nucleic acid technology or chemical synthesis. Also included in the present invention is the use of these proteins and nucleic acid molecules as well as antibodies and inhibitory compounds thereto as therapeutic compositions to protect animals from flea infestation as well as in other applications, such as those disclosed below.
[0036] Flea HMT and HNC proteins and nucleic acid molecules of the present invention have utility because they represent novel targets for anti-arthropod vaccines and chemotherapeutic drugs. The products and processes of the present invention are advantageous because they enable the inhibition of arthropod development, metamorphosis, feeding, digestion and/or reproduction processes that involve HMT and/or HNC proteins.
[0037] The head and nerve cord of the flea, including antennae, brain, corpora cardiacum, corpora allata, and subesophageal and abdominal ganglion tissues are of interest as such tissues are highly enriched for transcripts that encode neuronal and endocrine targets, as well as targets involved in chemosensory and mechanosensory reception. By sequencing cDNA fragments from a library enriched in flea head and nerve cord nucleic acid sequences (referred to herein as HNC nucleic acid sequences), genes, and their respective full-length coding regions, integrally involved with flea neuronal and endocrine function are identified. Once identified, these genes can be further characterized and specific interference strategies are designed. As such, flea HNC proteins and nucleic acid molecules of the present invention have utility because they represent novel targets for anti-arthropod vaccines and chemotherapeutic drugs.
[0038] Blood-feeding insects such as fleas ingest large quantities of blood relative to their body weight and, as such, are adapted to reduce the volume of the ingested blood meal through the rapid elimination of water. In addition, the concentrations of sodium, potassium, and chloride ions in the blood meal are greater than in the hemolymph of fleas, necessitating the excretion of excessive amounts of these ions. The active transport of these ions from the hemolymph into the lumens of the Malpighian tubules and the hindgut drives the passive transport of water and other hemolymph contents into these organs as well. While passing through these organs, waste products from the hemolymph are excreted and needed nutrients, water, and salts are reabsorbed. As such, interfering with these essential processes is an important strategy for developing a product for controlling flea populations. By sequencing cDNA fragments from a library enriched in hindgut and Malpighian tubule nucleic acid sequences (referred to herein as HMT nucleic acid sequences), genes integrally involved with these processes, and their respective full-length coding regions, are identified. Once identified, these genes are further characterized and specific interference strategies can be designed. As such, flea HMT proteins and nucleic acid molecules of the present invention have utility because they represent novel targets for anti-arthropod vaccines and chemotherapeutic drugs.
[0039] One embodiment of the present invention is an isolated protein that includes a flea HMT and/or HNC protein. It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, a protein, a nucleic acid molecule, an antibody and a therapeutic composition refers to “one or more” or “at least one” protein, nucleic acid molecule, antibody and therapeutic composition respectively. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. It is also to be noted that the terms “comprising”, “including”, and “having” can be used interchangeably. According to the present invention, an isolated, or biologically pure, protein, is a protein that has been removed from its natural milieu. As such, “isolated” and “biologically pure” do not necessarily reflect the extent to which the protein has been purified. An isolated protein of the present invention can be obtained from its natural source, can be produced using recombinant DNA technology, or can be produced by chemical synthesis.
[0040] As used herein, isolated flea HMT and/or HNC proteins of the present invention can be full-length proteins or any homologue of such proteins. An isolated protein of the present invention, including a homologue, can be identified in a straight-forward manner by the protein's ability to elicit an immune response against a flea HMT and/or HNC protein or by the protein's HMT and/or HNC activity. Examples of flea HMT and HNC homologue proteins include flea HMT and HNC proteins in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl inositol) such that the homologue includes at least one epitope capable of eliciting an immune response against a flea HMT or HNC protein, and/or of binding to an antibody directed against a flea HMT or HNC protein. That is, when the homologue is administered to an animal as an immunogen, using techniques known to those skilled in the art, the animal will produce an immune response against at least one epitope of a natural flea HMT or HNC protein. The ability of a protein to effect an immune response can be measured using techniques known to those skilled in the art. As used herein, the term “epitope” refers to the smallest portion of a protein or other antigen capable of selectively binding to the antigen binding site of an antibody or a T cell receptor. It is well accepted by those skilled in the art that the minimal size of a protein epitope is about four to six amino acids. As is appreciated by those skilled in the art, an epitope can include amino acids that naturally are contiguous to each other as well as amino acids that, due to the tertiary structure of the natural protein, are in sufficiently close proximity to form an epitope. According to the present invention, an epitope includes a portion of a protein comprising at least about 4 amino acids, at least about 5 amino acids, at least about 6 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 25 amino acids, at least about 30 amino acids, at least about 35 amino acids, at least about 40 amino acids or at least about 50 amino acids in length.
[0041] In one embodiment of the present invention a flea homologue protein has HMT or HNC activity, i.e. the homologue exhibits an activity similar to its natural counterpart. Examples of such activities are disclosed herein; e.g., all. Methods to detect and measure such activities are known to those skilled in the art. Examples of such activities are disclosed herein; e.g. allantoinase, chitin-binding protein, sodium/potassium ATPase, ligand-gated chloride channel, ANON/23DA, malvolio, odorant binding protein-like protein, N-methyl-D-aspartate receptor associated protein, chemical sense related lipophilic ligand binding protein, Sodium/Hydrogen Transporter-like protein, a Chloride Intracellular Channel-like protein, aPeritrophin-like protein, aPeritrophin-like protein, aPeritrophin-like protein, a synaptic vesicle 2B-like protein, a voltage-gated Chloride-like protein, an anoxia upregulated protein-like protein, and a neuroendocrine specific 7B2-like protein.
[0042] Flea HMT and/or HNC homologue proteins can be the result of natural allelic variation or natural mutation. Flea HMT and/or HNC protein homologues of the present invention can also be produced using techniques known in the art including, but not limited to, direct modifications to the protein or modifications to the gene encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.
[0043] Flea HMT and HNC proteins of the present invention are encoded by flea HMT and HNC nucleic acid molecules, respectively. As used herein, flea HMT and HNC nucleic acid molecules include nucleic acid sequences related to natural flea HMT and HNC genes, and, preferably, to
[0044] One embodiment of the present invention is a
[0045] Translation of SEQ ID NO:1, the coding strand of nCfALN
[0046] Translation of SEQ ID NO:7, the coding strand of nCfCBP
[0047] Translation of SEQ ID NO:13, the coding strand of nCfNKAB
[0048] Translation of SEQ ID NO:19, the coding strand of nCfLGIC
[0049] Translation of SEQ ID NO:25, the coding strand of nCfANON
[0050] Translation of SEQ ID NO:31, the coding strand of nCfMALV
[0051] Translation of SEQ ID NO:37, the coding strand of nCfOSD
[0052] Translation of SEQ ID NO:43, the coding strand ofNMDA
[0053] Translation of SEQ ID NO:153, the coding strand of nCfCLBP1A
[0054] Translation of SEQ ID NO:162, the coding strand of nCfCLBP2A
[0055] Translation of SEQ ID NO:1861, the coding strand of nCfLGIC
[0056] Translation of SEQ ID NO:1867, the coding strand of nCfNAH
[0057] Translation of SEQ ID NO:1872, the coding strand of nCfCLIC
[0058] Translation of SEQ ID NO:1882, the coding strand of nCfPL2
[0059] Translation of SEQ ID NO:1887, the coding strand of nCfPL3
[0060] Translation of SEQ ID NO:1896, the coding strand of nCfPL4
[0061] Translation of SEQ ID NO:1901, the coding strand of nCfSVP
[0062] Translation of SEQ ID NO:1914, the coding strand of nCfVGCC
[0063] Translation of SEQ ID NO:1919, the coding strand of nCfAUP
[0064] Translation of SEQ ID NO:1924, the coding strand of nCf7B2
[0065] Table I represents a variety of flea HNC nucleic acid molecules of the present invention. Also cited in Table I are nucleic acid molecules from other organisms which share the closest sequence identity with the cited HNC sequences of the present invention, as determined by submitting each HNC sequence for a search through the National Center for Biotechnology Information (NCBI), National Library of Medicine, National Institute of Health, Baltimore, Md., using the BLAST network. This database includes SwissProt+PIR+SPupdate+GenPept+GPUpdate+PDB databases. The search was conducted using the xBLAST function using default parameters.
TABLE I SEQ ID NO: Name Genbank Homology Organism 63 2096-46 ATPase 6 64 2098-25 ATP synthase delta chain 65 2098-34 F1-ATPase epsilon-subunit 66 2110-19 ATP synthase beta subunit 67 2113-15 ATP synthase delta chain, 68 2180-31 ATP synthase alpha subunit precursor 69 2224-50 oligomysin sensitivity conferring protein 70 2116-51 cysteine dioxygenase 71 2116-55 pyrroline-5-carboxylate dehydrogenase (P5CDh) 72 2124-17 AMP deaminase 73 2138-38 ubiquitin 74 2184-59 manganese superoxide dismutase 75 2096-24 muscle LIM protein 1 76 2140-53 F25H5.1a 77 2176-41 Frazzled 78 2223-11 LIMm domain-containing protein 79 2223-53 deleted in split hand/split foot 1 (DSS1) 80 2225-28 stranded-at-second 81 2099-61 histone H3 82 2114-21 STE12 83 2117-4 Rad51 homolog 84 2138-46 heat shock protein p27 85 2182-37 heat shock protein 70 86 2211-32 BTB-II protein domain gene 87 2223-7 heat shock protein 88 2224-17 heat shock protein 86 89 2225-16 POU domain protein 90 2225-18 nucleolin 91 2212-85 thyroid hormone receptor-associated protein complex component TRAP220 92 2211-21 T03D8.3 93 2223-67 hepatoma derived growth factor (HDGF) 94 2225-61 tyrosine hydroxylase type 1 (neuronal form) 95 2097-7 sarco/endoplasmic reticulum-type Ca-2+-ATPase 96 2098-27 calcium-transporting ATPase 97 2099-19 calcium channel alpha-1 subunit 98 2120-5 P-type voltage-gated calcium channel alpha 1 subunit homolog 99 2124-2 sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) 100 2182-43 sulfonylurea receptor 2b 101 2223-18 Sodium-Potassium-Chloride cotransporter 102 2223-63 sarco/endoplasmic reticulum-type Ca2(+)-ATPase 103 2224-13 similar to ABC transporters 104 2098-3 Camguk 105 2101-9 UNC-89 106 2132-31 arginine kinase 107 2141-51 casein kinase-II beta 108 2178-18 diacylglycerol kinase eta Cricetinae 109 2180-32 retinoid- and fatty acid-binding glycoprotein 110 2137-23 vitellogenin 111 2144-14 nuclear localization signal spot 1 112 2212-13 putative n- terminal acetyltransferase 113 2212-27 clathrin associated protein AP47 114 2223-28 O1 chloroquine-resistance protein 115 2224-14 vitellogenin 116 2224-15 antigen NY-CO-3 117 2225-24 carbonic anhydrase 118 2225-58 yk500f6.3 119 2225-76 unknown 120 2224-86 BmP109 (cerebroside sulfate activator protein family) 121 2225-23 intersectin 122 2170-16 chemical-sense-related lipophilic-ligand-binding protein 123 2176-2 olfactory receptor protein 2.4 124 2212-63 olfactory receptor 125 2224-77 inner mitochondrial membrane translocase Tim23 126 2225-12 sodium-dependent multi-vitamin transporter 127 2225-42 ribophorin I 128 2101-59 phosphate carrier protein 129 2132-38 proteinase inhibitor 130 2174-72 HE4 protein 131 2211-48 spermatogenic cell/sperm-associated Tat-binding homologue 132 2110-23 Gcap1 gene product 133 2116-64 toll protein 134 2124-3 tuberin (TSC2) gene 135 2178-55 RAS-like protein 136 2223-35 Rho1 gene product 137 2224-82 paxillin 138 2225-44 adenylyl cyclase-associated protein (CAP) 139 2225-80 adenylate kinase 140 2110-52 hydroxyproline-rich glycoprotein 141 2115-49 mitogen inducible gene mig-2 142 2116-5 F52H3.5 143 2172-89 12D3 antigen 144 2178-20 frameshift 145 2178-81 KIAA0066 146 2182-16 Y57G11C.4 147 2182-53 C16C10.5 148 2211-8 Unknown 149 2211-31 hopothetical protein 150 2223-54 ORF YNL207w 151 2224-94 14.3 kDa perchloric acid soluble protein 152 2225-36 EST clone 1719 2228-2 BIGH3 1720 2228-5 H protein 1721 2228-8 ubiquinol-cytochrome c reductase 1722 2228-11 similar to mitochondrial ATPase inhibitors 1723 2228-16 Putative enzyme 1724 2228-18 Ribosomal protein L7A Drosophila 1725 2228-22 Troponin-I wings up A Drosophila 1726 2228-25 tls gene product 1727 2228-27 YCR521 gene product 1728 2228-28 putative transport system permease protein 1729 2228-32 SapA protein 1730 2228-34 Putative protein 1731 2228-37 Ada 1732 2228-39 Titin 1733 2228-42 adenylosuccinate synthetase 1734 2228-43 transfer RNA-Ala synthetase 1735 2228-44 C4 zinc finger DNA-binding protein Drosophila 1736 2228-48 heme A: farnesyltransferase 1737 2228-51 URF 4L (aa 1-96) Drosophila 1738 2228-53 DOLICHOL-PHOSPHATE MANNOSYLTRANSFERASE 1739 2228-58 troponin-T Drosophila 1740 2228-59 protein disulfide isomerase Drosophila 1741 2228-63 orf, hypothetical protein 1742 2228-66 ilvl polypeptide 1743 2228-68 orf, hypothetical protein 1744 2228-72 Respiratory nitrate reductase 1 alpha chain 1745 2228-77 homolog of virulence factor 1746 2228-84 ORF o164 1747 2228-91 nuclear protein E3-3 orf1 1748 2245-66 Troponin C Drosophila 1749 2245-70 Predicted secreted protein 1750 2245-72 Cytochrome C-1 1751 2245-75 rpoB 1752 2245-78 sarco(endo) plasmic reticulum-type calcium ATPase 1753 2246-31 Ras-related GTP-binding protein 1754 2246-57 Similar to inositol 1,4,5-triphosphate receptor 1755 2246-61 reverse transcriptase-like protein 1756 2247-13 polyprotein Drosophila 1757 2247-14 ORF2 for putative reverse transcriptase Drosophila 1758 2247-42 Asparaginyl tRNA Synthetase 1759 2247-44 calcium binding protein Drosophila 1760 2247-58 similar to Fibronectin type III domain 1761 2247-62 reverse transcriptase Drosophila 1762 2247-65 gag-like protein 1763 2247-79 L-3-phosphoserine phosphatase 1764 2247-80 esterase E4 1765 2247-89 Similar to aldehyde dehydrogenase 1766 2248-76 O-44 protein Rattus sp. 1767 2248-85 cDNA isolated for this protein using a monoclonal antibody directed against the p27k prosomal protein 1768 2249-3 Projectin Drosophila 1769 2249-5 ORF_ID:o312#14 1770 2249-9 Heat shock protein 60 1771 2249-11 enigma protein 1772 2249-12 alpha, alpha-trehalose glucohydrolase 1773 2249-13 small GTP binding protein Drosophila 1774 2249-14 Spermidine/putrescine transport system permease 1775 2249-19 nueroendocrine-specific protein C 1776 2249-21 a-agglutinin core subunit 1777 2249-24 KIAA0337 1778 2249-34 su(wa) protein Drosophila 1779 2249-42 regulator of kdp operon 1780 2249-59 No definition line found 1781 2249-60 proline oxidase Drosophila 1782 2249-62 Formate acetyltransferase 1783 2249-70 similar to HECT-domain 1784 2249-75 PHOSPHORIBOSYLFORMYLGLYCINAM IDINE CYCLO-LIGASE 1785 2249-77 Hypothetical 38.5 kd protein in agal-mtr intergenic region precursor 1786 2249-85 D4L Variola virus 1787 2249-87 similar to isocitrate dehydrogenase 1788 2250-6 Fii (head-tail joining; 117) 1789 2250-7 possible NAGC-like transcriptional regulator 1790 2250-10 cysteine string protein 1791 2250-13 Tol B protein 1792 2250-14 6-phosphogluconate dehydratase 1793 2250-15 6-phosphogluconate dehydratase 1794 2250-22 PSST subunit of the NADH: ubiquinone oxidoreductase 1795 2250-30 sol i 3 antigen 1796 2250-36 predicted using Genefinder; similar to tRNA synthetases class I (E and Q 1797 2250-37 PNP 1798 2250-42 ORF_ID:o331#2 1799 2250-44 Extensin 1800 2250-47 ORF o654 1801 2250-48 Gcap1 gene product 1802 2250-52 similar to human MLH1 on chromosome 3p21 1803 2250-53 Hypothetical 27.6 kd protein in hpt-panD intergenic region. 1804 2250-58 UmuC protein 1805 2250-61 dJ134E15.1 (Blimp-1 1806 2250-63 ribosomal protein L23-related product homolog 1807 2250-65 hypothetical protein MJ1143 1808 2250-68 HI0025 homolog 1809 2250-77 R34094_1 1810 2250-78 erythrocyte binding protein 1811 2250-79 fosmidomycin resistance protein 1812 2250-81 cyclophilin 1 Drosophila 1813 2250-83 putative glutamine synthetase 1814 2251-3 J (tail:host specificity; 1132) 1815 2251-5 Molybdopterin biosynthesis MoeB protein 1816 2251-6 Fo-ATP synthase subunit b Drosophila 1817 2251-9 citrate lyase alpha chain 1818 2251-10 cuticle protein ACP65A Drosophila 1819 2251-13 H repeat-associated protein in rhsC 3'region (orf-h3 1820 2251-20 glycine-rich protein 1821 2251-23 2-oxoglutarate dehydrogenase precursor 1822 2251-29 NFX1 1823 2251-32 ebgR product, repressor 1824 2251-41 neural protein Drosophila 1825 2251-45 similar to unidentified ORF 1826 2251-46 NADH:ubiquinone oxidoreductase b17.2 subunit 1827 2251-49 tyrosine kinase Drosophila 1828 2251-50 coded for by 1829 2251-57 H (tail component; 853) 1830 2251-60 Lysyl tRNA Synthetase Drosophila 1831 2251-62 7,8-diamino-pelargonic acid aminotransferase 1832 2251-64 actin related protein Drosophila 1833 2252-6 discs-large tumor suppressor Drosophila 1834 2252-16 S-adenosylmethionine decarboxylase 1835 2252-17 F52H3.5 1836 2252-21 translationally controlled tumor protein 1837 2252-31 GTP binding protein 1838 2252-34 mitochondrial porin transcript 1 Drosophila 1839 2252-38 cuticle protein 1840 2252-39 Similarity to Rat CD63 antigen 1841 2252-41 similar to S. cerevisiae Lpg20p 1842 2252-48 cut E 1843 2252-61 Histone H3 1844 2252-66 ea10 (ssb; 122) 1845 2252-71 Mao C protein 1846 2252-72 miniparomyosin Drosophila 1847 2252-73 pherophorin-S 1848 2252-80 cyclophilin 1849 2252-84 alternate gene name yhhG 1850 2222-20 nucleoporin Nup98 rat 1851 2222-21 hypothetical protein 1852 2222-36 ribosomal protein S11 human 1853 2222-39 hypothetical protein PFB0315w 1854 2222-50 serine/threonine-specific protein k. 1855 2222-58 hypothetical protein C25E10.9 1856 2222-64 transporting ATP synthase bovine 1857 2222-94 tricarboxylate carrier rat 1858 2218-95 anoxia upregulated protein
[0066] Table II represents a variety of flea HMT nucleic acid molecules of the present invention. Also cited in Table II are nucleic acid molecules from other organisms which share the closest sequence identity with the cited HMT sequences of the present invention, as determined by a search through the BLAST network as described above.
TABLE II SEQ ID NO: Name GenBank Homology Organism 171 2094-23 mitochondrian ATP synthase, alpha subunit 172 2104-20 mitochondrial ATP synthase 173 2105-14 ATP synthase gamma-subunit 174 2167-72 oligomysin sensitivity conferring protein 175 2179-20 ATPase 6 176 2193-60 ATP synthase subunit B 177 2229-41 ATP synthase alpha subunit 178 2231-35 9 kD basic protein 179 2231-47 ATP synthase alpha-subunit 180 2232-95 mitochondrial ATP synthase subunit 9 181 2084-56 Late embryogenesis abundant protein 182 2084-36 TGF-beta masking protein/stranded at second 183 2086-2 Argonaute protein 184 2196-92 like Drosophila HMPB homeotic proboscipedia protein 185 2092-27 DMDHEM2 186 2094-21 SeID protein 187 2106-11 Unr 188 2231-15 cno (canoe) 189 2230-79 ALR homologue 190 2232-42 saxophone serine-threonine kinase receptor 191 2232-68 selenophosphate synthetase 192 2088-11 MMTAX107, TAX responsive element binding protein 193 2089-2 cs Dna J-1 194 2090-7 Lethal (2) TID 195 2102-33 monocytic leukaemia zinc finger protein 196 2105-26 orf1 5′of EpoR 197 2106-6 contains similarity to EGF-1 198 2106-9 HSP70 protein 199 2084-60 82 kD heat shock protein 200 2108-59 PAR domain protein 201 2156-34 yk29g12.3 202 2161-17 segmentation protein 203 2162-28 heat shock protein 70, hsp70A2 204 2187-18 Heat shock protein 70 205 2173-77 Heat shock protein hsp70 206 2165-30 nucleolar protein 207 2165-59 contains similarity to C4-type zinc fingers 208 2177-80 zinc finger protein 209 2181-45 PAR domain protein 1 210 2185-9 Heat shock protein-70 211 2185-82 segmentation protein 212 2188-33 transcriptional repressor protein 213 2203-18 Mastermind 214 2205-82 high mobility group protein 1a 215 2230-26 DNA repair protein 216 2230-71 homologue of seven in absentia 217 2230-89 nuclear speckle-type protein, SPOP 218 2230-96 heat shock protein 219 2231-7 hypothetical protein 220 2231-38 Rad51 homolog 221 2231-81 DNA repair protein 222 2232-2 cellular nucleic acid binding protein 223 2234-63 heat shock protein 70 Trichoplusia ni 224 2232-77 actin-binding double-zinc-finger protein (abLIM) 225 2234-78 DNA-binding protein isoform I 226 2084-48 Allantoinase 227 2085-22 beta-glucuronidase 228 2094-24 prolidase = peptidaseD/imidopeptidase 229 2088-43 branched chain alpha-keto acid dehydrogenase E1-beta subunit 230 2086-29 3-hydroxyisobutyrate dehydrogenase 231 2088-5 Rab 5c protein 232 2095-17 cytochrome P-450 233 2102-16 carbamoyl phosphate synthetase II 234 2102-48 NADPH cytochrome P450 reductase 235 2104-15 branched chain alpha-keto acid dehydrogenase 236 2106-5 Metallothionein 237 2106-47 peroxidoxin-1 238 2107-17 tetracycline transporter-like protein 239 2107-58 allergen Bla g 5 (glutathione-S-transferase) 240 2156-58 HAL-3 homologue 241 2195-90 aminoacyclase-1 242 2171-55 NADPH-ferrihemoprotein reductase 243 2169-30 hypothetical protein Synechocystis sp 244 2169-52 insulin degrading enzyme 245 2177-64 3-hydroxyisobutyrate dehydrogenase 246 2181-69 Endonexin 247 2138-25 glutamate dehydrogenase 248 2230-28 glutathione -S-transferase 249 2191-8 lactase-phlorizin hydrolase 250 2193-52 cytochrome P450 251 2202-35 glutathione-S-transferase 252 2229-77 glutathione-S-transferase 253 2229-81 urate oxidase 254 2231-42 superoxide dismutase 255 2232-74 allergen Bla g 5 256 2234-42 glutathione reductase family 257 2087-8 cystic fibrosis transmembrane conductance regulator 258 2087-23 Nervous system antigen 2 259 2091-56 adenosine triphosphatase 260 2094-20 sodium pump, alpha suhbunit 261 2095-51 similar to Hrs 262 2103-24 N-methyl-D-aspartate receptor-associated protein 263 2105-55 inward rectifying K channel 264 2105-63 EF-hand Ca2+ binding protein p22 265 2106-62 Dents disease candidate gene product 266 2167-50 PKD1 (polycystic kidney disease 1) 267 2185-37 copper-transporting ATPase 268 2193-29 TrkG Potassium transport protein 269 2195-33 silicon transporter 270 2202-16 similarity to human sulfate anion transporter 271 2230-2 sulfate transporter 272 2230-69 mitochondrial porin 273 2231-22 muscarinic acetylcholine receptor 274 2231-24 p97 subunit of 15S Mg(2+)- ATPase 275 2231-32 anion transporting ATPase 276 2231-70 sulfate permease 277 2231-94 putative Na/H exchanger 278 2233-6 plasma membrane Ca2+-ATPase 2 279 2233-24 chloride channel gene, CLIC2 280 2085-61 beta-type protein kinase C 281 2089-20 cGMP-dependent protein kinase 282 2092-12 Btk 283 2093-64 Receptor-like protein tyrosine phosphatase 284 2095-31 frt (fms-related tyrosine kinase gene) 285 2094-58 casein kinase II beta 286 2103-54 ORF YGL084c 287 2106-42 protein phosphatase epsilon subunit 288 2156-5 serine/threonine kinase 289 2157-95 cGMP-dependent protein kinase 290 2165-80 ABL gene product 291 2165-63 diadenosine tetraphosphatase 292 2167-17 adenylate cyclase 293 2177-44 serine/threonine kinase 294 2188-16 weakly similar to serine/threonine kinase 295 2191-60 carbohydrate kinase, pfkB family 296 2195-22 protein kinase 297 2196-30 calcium-dependent protein kinase 298 2205-83 protein kinase/endoribonulcease (IRE1) 299 2205-87 receptor tyrosine phosphatase 300 2229-11 magnesium-dependent calcium inhibitable phosphatase 301 2229-29 phosphoglycerate kinase 302 2229-74 pyruvate kinase 303 2230-55 serine/threonine specific protein phosphatase 4 304 2230-57 stress activated MAP kinase kinase 3 305 2231-64 alkaline phosphatase 306 2231-91 olynucleotide phosphorylase 307 2232-43 protein kinase PkwA 308 2234-94 serine/threonine kinase ULK1 309 2085-18 Pyridoxamine phosphate oxidase 310 2094-13 sphingomyelin phosphodiesterase 311 2105-47 apolipoprotein E receptor 2 312 2092-38 squalene synthetase 313 2094-25 fatty acid synthetase 314 2089-32 coproporphyrinogen oxidase 315 2085-46 HADHB mitochondrial trifunctional protein beta subunit 316 2104-56 pyridoxal kinase 317 2107-30 Phosphomevalonate kinase 318 2154-70 very-long chain acyl-CoA dehydrogenase 319 2191-85 stearyl-CoA desaturase 320 2192-44 Very-long-chain Acyl-CoA dehydrogenase 321 2195-55 Similar to LDL receptor-related protein 322 2229-82 lipase-3 323 2231-59 Phosphatidylethanolamine-binding protein 324 2233-25 similarity to yeast ethanolaminephosphotransferase 325 2233-41 cellular retinoic acid binding protein (mCRABP) 326 2087-61 I allergen 327 2087-41 chloroquine resistance candidate protein 328 2089-51 Xenopus Bf B 329 2086-58 repeat organellar protein 330 2090-45 heat shock cognate protein 331 2104-23 40 kDa heat shock chaperone protein Deinococcus 332 2107-26 Luciferase 333 2162-46 F20D1.9 334 2162-49 PKR inhibitor P58 335 2162-93 GroES homologue Ricketsia 336 2171-46 NH2 terminus uncertain 337 2089-10 beta adaptin 338 2229-24 non-functional folate binding protein 339 2229-25 calmodulin B 340 2229-31 putative T1/ST2receptor binding protein 341 2229-36 alpha-crystallin cognate protein 25 342 2229-40 Defensin 343 2229-86 glutamate-ammonia ligase 344 2231-49 melanoma-associated antigen ME491 345 2231-76 histone C 346 2232-65 translationally controlled tumor protein 347 2232-84 Apyrase 348 2232-85 KIAA0124 349 2233-59 Glutamine-dependent carbamoyl-phosphate synthase 350 2233-86 ANG12 precursor 351 2234-11 tissue specific secretory protein 352 2234-76 methionine adenosyltransferase 353 2089-13 Synaptic vessicle protein 2 form B 354 2159-52 glycoprotein 56 355 2084-6 CLN3; homologue of the gene underlying Batten disease 356 2085-10 Amphiphysin 357 2156-39 glycoprotein 55 358 2104-59 Transmembrane transporter 359 2105-9 insect intestinal mucin II Trichoplusia ni 360 2106-14 kinesin-like protein 361 2107-45 Lazarillo precursor 362 2156-3 clathrin-associated protein 363 2161-46 neural variant mena + protein 364 2171-92 Malvolio 365 2175-18 homolog of SYT - synaptotagmin 366 2177-10 GABA receptor subunit (Rdl) 367 2181-10 neurexin IV 368 2191-92 synaptic vessicle protein 2B 369 2229-18 Synaptic vessicle protein 2A 370 2194-38 gamma-subunit of mouse nerve growth factor 371 2230-60 lin-7-C 372 2230-81 PDZ domain protein 373 2234-5 Gcap1 gene product 374 2234-55 Gcap1 gene product 375 2234-71 Gcap1 gene product 376 2085-34 Liver-specific transport protein 377 2087-15 polyspecific organic cation transporter 378 2204-80 transmembrane transporter 379 2093-39 liver-specific transport protein 380 2093-46 similar to monocarboxylate transporter family 381 2092-22 similar to matrin F/G 382 2103-50 Unknown 383 2103-51 organic cation transporter 384 2197-35 renal organic cation transporter 385 2156-17 sulfate anion transporter 386 2166-84 LX1 387 2167-94 MCT (monocarboxylate transporter) 388 2196-83 renal organic cation transporter 389 2229-83 similarity to monocarboxylate transporter 1 390 2231-89 Golgi 4-transmembrane spanning transporter MTP 391 2158-8 phosphate carrier protein 392 2085-14 ADP/ATP translocase 393 2085-17 Na+-dependent inorganic phosphatase cotransporter 394 2088-38 ADP/ATP translocase 395 2092-50 ADP/ATP translocase 396 2104-21 Na(+)-dependent inorganic phosphate cotransporter 397 2121-55 phosphate carrier protein 398 2105-64 phosphate carrier protein 399 2102-6 ZK512.6 400 2108-27 mitochondrial phosphate carrier protein 401 2194-63 mitochondrial phosphate transporter 402 2196-14 phosphate/triose-phosphate translocator precursor 403 2204-11 EST clone 404 2085-16 Chymotrypsin I 405 2085-54 Chymotrypsin II 406 2086-12 Plasminogen 407 2086-18 Trypsin eta 408 2090-21 Trypsin 409 2092-15 Alp1 410 2102-11 vitellin-degrading protease 411 2102-17 Chymotrypsin II 412 2102-51 chymotrypsin-like protease 413 2103-31 Beta trypsin 414 2107-22 Factor IX 415 2108-29 Trypsin 416 2157-15 Trypsin 417 2160-34 Aminopeptidase Synechocystis 418 2160-36 E01G6.1 419 2103-62 plasminogen activator inhibitor 2 420 2167-36 factor IX 421 2167-67 Alp1 422 2169-51 Trypsin 423 2181-27 Chymotrypsin BII 424 2185-69 plasma prekallikrein 425 2187-20 pre-procathepsin L 426 2188-45 vitellin-degrading protease 427 2192-91 late trypsin precourser quinquefasciatus 428 2196-10 SPC2 429 2196-88 Trypsin 430 2204-9 carnitine/choline acetyltransferase 431 2229-7 iota trypsin 432 2229-22 Trypsin 433 2229-89 Trypsin 434 2229-94 late trypsin precourser quinquefasciatus 435 2230-59 Chymotrypsin 1 436 2230-67 carboxypeptidase A 437 2231-62 aminopeptidase N 438 2231-74 limulus factor C serine protease 439 2232-15 cysteine proteinase 440 2232-25 Carboxypeptidase 441 2232-33 putative aspartic protease 442 2233-46 aminopeptidase N 443 2233-85 chymotrypsin 1 444 2233-90 Trypsin 445 2233-94 preprechymotrypsin 1 446 2234-29 chymotrypsin-like protease precursor 447 2234-58 Putative 448 2234-61 carboxylesterase precursor 449 2234-68 serine protease inhibitor I 450 2084-35 Integral membrane protein 451 2086-45 similar to beta-ureidopropionase of Rat 452 2087-54 Cyclin 453 2088-22 Esp 8 454 2091-16 contains similarity to EGF-like domains 455 2091-29 multiple exostosis-like protein 456 2091-30 apoptosis 1 inhibitor 457 2092-33 KIAA0023 (putitive oncogene) 458 2095-35 G coupled receptor 459 2095-3 Go (heterotrimeric guanyl nucleotide binding protein alpha subunit) 460 2085-4 gp 150 protein 461 2103-28 leukotriene A4 hydrolase Rattus sp. 462 2105-62 putitive orf 463 2107-6 activator protein 464 2107-28 platelet-endothelial tetraspan antigen 3 465 2189-3 oligopeptidase A (prlC) 466 2156-54 fibroblast growth factor receptor 467 2160-92 contains similarity to EGF-like domains 468 2160-65 weak similarity to the drosophila hyperplastic disc protein 469 2165-53 inositol triphosphate receptor 470 2166-22 placental protein 11 471 2166-92 elongation factor 1 alpha-like 472 2181-34 DSch 473 2192-65 STAM, signal transducing adaptor molecule 474 2194-24 ATPases associated with various cellular activities (AAA family) 475 2196-75 similar to cell division control protein 476 2230-38 EST clone 477 2230-39 NTPase 478 2230-66 adenylyl cyclase aggregation protein 479 2230-80 sphingomyelin phosphodiesterase 480 2231-29 nuclear antigen H731 481 2231-40 suppressor of actin mutation 2 482 2231-66 DET1 483 2232-7 Calreticulin 484 2232-38 activator protein 485 2232-69 ornithine decarboxylase 486 2233-32 similar bHLH-PAS 487 2233-45 rab1 488 2234-2 C10A gene product 489 2234-72 QM homolog 490 2084-17 Integral membrane protein Herpesvirus-2 491 2091-4 endomembrane protien EMP70 precourser isolog 492 2102-45 Ylr251wp 493 2162-68 220 kDa silk protein 494 2160-47 precursor HT7 protein 495 2161-12 peritrophin 95 precourser 496 2161-15 yk86g11.5 497 2171-12 51A surface protein 498 2173-18 hypothetical - mitochondrial membrane transport protein 499 2087-32 est sequence 500 2091-19 Similar to P. aeruginosa hypothetical protein 501 2192-86 tyrosine kinase 502 2086-42 M04B2.4 503 2088-16 glycoprotein 330 504 2088-39 EST sequence 505 2088-57 Yer 126cp 506 2089-25 similar to protein YKL166 507 2090-3 EST sequence 508 2090-53 EST sequence 509 2095-20 Chloroplast ORF 510 2102-28 similar to S. cerevisiae hypothetical protein YKL166 511 2102-55 D1054.3 512 2102-58 ZC513.5 gene product 513 2105-44 E 1087 protein 514 2109-24 F11C1.5 515 2154-21 disulfide-like protein 516 2156-6 ZK470.1 517 2156-18 BIIIA3 518 2156-27 AFR1 519 2165-94 COS41.8 520 2167-65 EST sequence, function unknown 521 2171-93 KIAA0160 522 2175-45 ORF YJR83.18 523 2185-66 rps4 524 2195-40 C27C12.4 525 2196-20 glycoprotein A 526 2205-89 BKRF1 encodes EBNA-1 protein Epstein Barr virus 527 2229-19 D4L Variola virus 528 2230-35 KIAA0747 529 2231-8 I3 530 2231-78 unknown protein 531 2232-49 Similarity to Yeast hypothetical 52.9 KD protein 532 2232-52 tetratricopeptide repeat protein (tpr2) 533 2233-5 similar to Saccharomyces cerevisiae SCD6 protein 534 2233-22 cDNA EST yk486b9.3 535 2233-93 CDC27Dm 536 2084-34 Immune suppressor/V-ATPase 115 kDa subunit 537 2086-30 V-ATPase A-subunit 538 2087-45 H+ATPase 539 2088-55 40-kDa-V-ATPase subunit 540 2088-62 vacuolar ATPase subunit A 541 2091-26 proton-ATPase-like protein 542 2091-31 vacuolar ATPase subunit A 543 2092-20 vacuolar ATPase 115 kDa subunit 544 2095-18 similar to 54 kD subunit 545 2095-54 H (+)-transporting ATPase subunit B 546 2108-8 similar to subunit 547 2154-36 V-ATPase subunit E 548 2154-76 V-ATPase subunit A (new fragment) 549 2166-32 V-ATPase C subunit 550 2166-33 vacuolar (V-type) H(+)-ATPase B subunit Helicoverpa virescens 551 2166-90 beta subunit of ATPase 552 2161-5 ATPase I 553 2171-24 similar to V-ATPase 116 kd subunit 554 2169-82 V-ATPase subunit E 555 2187-36 V-ATPase membrane sector associated protein M8-9 556 2188-91 V-ATPase subunit A 557 2230-88 vacuolar ATPase G subunit 558 2232-61 V-ATPase subunit C 559 2086-52 Penelope transposable element ORF 560 2103-2 genome polyprotein gene product Plum pox virus 561 2106-8 pol protein Human T-cell lymphotropic virus type 2 562 2108-41 reverse transcriptase, Doc retroposon 563 2202-28 Polyprotein Hepatitis virus C 564 2165-95 DNA polymerase entomopoxvirus 565 2169-81 reverse transcriptase 566 2181-36 reverse transcriptase 1416 2240-4 alpha-L-fucosidase precursor 1417 2240-11 estrogen related receptor alpha 1418 2240-14 NADH: ubiquinone oxidoreductase 51-kD subunit 1419 2240-17 peritrophin 1 1420 2240-19 small GTPase rac1b 1421 2240-23 Symplekin 1422 2240-26 ribosomal protein L30 1423 2240-28 60S Ribosomal Protein RPL10A 1424 2240-29 KIN17 protein 1425 2240-31 eukaryotic initiation factor 4 gamma 1426 2240-38 ornithine decarboxylase antizyme 1427 2240-44 electron transfer flavoprotein 1428 2240-53 EST clone 1429 2240-55 glutathione reductase family 1430 2240-58 chymotrypsin-like serine protease 1431 2240-63 Ferritin subunit 1 1432 2240-64 vacuolar ATPase subunit B 1433 2240-66 chaperonin containing TCP-1 delta 1434 2240-70 1-acyl-glycerol-3-phosphate acyltransferase 1435 2240-71 EST clone AL021106 1436 2240-72 376aa long hypothetical dehydrogenase 1437 2240-77 chymotrypsin-like serine protease 1438 2240-80 EST clone 1439 2240-83 chymotrypsin-like serine protease 1440 2240-90 cytochrome P450 1441 2240-93 enhancer-trap-locus-1 1442 2240-94 glycerol-3-phosphate dehydrogenase 1443 2241-3 FS-H precourser 1444 2241-5 trypsin-like serine protease 1445 2241-7 myospheroid protein 1446 2241-10 Sam50 1447 2241-12 NADH dehydrogenase subunit 2 1448 2241-15 putative protein 1449 2241-16 contains EGF-like repeats 1450 2241-20 Gcap1 gene product 1451 2241-25 Na(+)-dependent inorganic phosphate cotransporter 1452 2241-31 D4L Variola virus 1453 2241-36 plenty-of-prolines-101; POP101; SH3-philo- protein 1454 2241-40 EF-1-alpha 1455 2241-44 F1-ATP synthase epsilon-subunit 1456 2241-54 ribosomal protein S28 1457 2241-55 Y-box protein 1458 2241-56 short-chain alcohol dehydrogenase 1459 2241-59 contains 3 cysteine rich repeats 1460 2241-60 muscle type phosphofructokinase 1461 2241-61 Heat shock protein 82 1462 2241-65 chymotrypsin-like protease 1463 2241-66 Oligosaccharyltransferase subunit 1464 2241-70 EST clone 1465 2241-72 failed axon connections protein 1466 2241-74 Enolase 1467 2241-78 multiple exostosis 2 protein 1468 2241-80 Protein on Ecdysone Puffs 1469 2241-82 paramyosin 1470 2241-83 beta-tubulin 1471 2241-84 natural killer cell enhancing factor 1472 2241-86 similar to MYOTUBULARIN-RELATED PROTEIN 1473 2241-87 Renin 1474 2241-90 Myophilin 1475 2243-10 alpha-actinin 1476 2243-11 monocarboxylate transporter 1477 2243-13 yk278a10.3 1478 2243-15 selenium donor protein 1479 2243-18 acetyl-CoA synthetase 1480 2243-20 cytochrome P450 CYP12A3 1481 2243-22 NADH dehydrogenase subunit 4 1482 2243-27 Polyubiquitin 1483 2243-28 Moesin 1484 2243-31 QM protein 1485 2243-32 Sec23 protein 1486 2243-37 truncated protein 1487 2243-38 Projectin 1488 2243-39 Unknown 1489 2243-41 similar to enoyl-CoA hydratase 1490 2243-45 similar to dehydrogenase 1491 2243-46 trypsin-like serine protease 1492 2243-48 Merlin 1493 2243-52 GTP-specific succinyl-CoA synthetase beta subunit 1494 2243-53 sod protein (superoxide dismutase) 1495 2243-54 trypsin-like serine protease 1496 2243-61 chymotrypsin-like serine protease 1497 2243-66 Tag B 1498 2243-67 hypothetical protien 1499 2243-68 heat shock cognate protein 70 Trichoplusia ni 1500 2243-72 TRIP-1 homologue 1501 2243-73 cytosolic NADP-dependent isocitrate dehydrogenase 1502 2243-86 progesterone-induced protein 1503 2243-87 Bmsqd-2 1504 2243-91 sodium/iodide symporter 1505 2243-92 ORF2 1506 2243-94 lysosomal beta-galactosidase 1507 2244-12 tropomyosin isoform 127 1508 2244-19 KIAA0181 1509 2244-23 plasma membrane calcium ATPase isoform 1 1510 2244-29 NADH dehydrogenase 1511 2244-44 glutamate dehydrogenase 1512 2244-54 spliceosomal protein 1513 2244-59 ciliary body glutathione peroxidase 1514 2244-61 pyridoxal-phoshate-dependent aminotransferases 1515 2244-64 Unknown 1516 2244-69 trypsin-like serine protease 1517 2244-71 peritrophin 1 1518 2244-75 NADH dehydrogenase subunit 5 1519 2244-84 microsomal epoxide hydrolase 1520 2244-86 C54G7.2 gene product 1521 2244-91 Aminopeptidase N 1522 2253-2 cytochrome C oxidase 1523 2253-13 Initiation factor 5A 1524 2253-14 protein phosphatase type 2A catalytic subunit 1525 2253-16 myosin light chain 2 1526 2253-18 cDNA EST yk462d1.5 1527 2253-19 ribosomal protein S10 1528 2253-24 aspartyl(asparaginyl)beta-hydroxylase, HAAH 1529 2253-27 larval and adult myosin heavy chain 1530 2253-33 nervous system antigen 2 1531 2253-36 dJ366N23.2 1532 2253-40 hrp48.1 1533 2253-42 ZnT-1 1534 2253-43 aminopeptidase N 1535 2253-56 Profilin 1536 2253-59 T26A5. 1537 2253-68 NADH-ubiquinone oxidoreductase 42 kDa subunit 1538 2253-78 glycine-rich protein 1539 2253-81 5′-nucleotidase 1540 2253-86 glutathione S-transferase 1541 2253-87 ferritin subunit 1 1542 2253-92 myosin light chain 2 1543 2253-94 xylose-proton symport 1544 2254-4 mature-parasite-infected erythrocyte surface antigen 1545 2254-6 Fo-ATP synthase subunit b 1546 2254-13 similar to protein 2 1547 2254-17 CLN3 protein 1548 2254-21 YbgG 1549 2254-25 peroxisomal protein Synechocystis sp 1550 2254-27 Glutaminase 1551 2254-30 tartan protein 1552 2254-33 leucine zipper-EF-hand containing transmembrane protein 1 1553 2254-39 similar to helicase 1554 2254-43 muscle myosin heavy chain 1555 2254-45 putative nicotinate phosphoribosyltransferase 1556 2254-51 60S ribosomal protein 1557 2254-54 small nuclear riboprotein Sm-D 1558 2254-55 nucleoside diphosphate kinase 1559 2254-60 serine protease 1560 2254-63 myospheroid protein 1561 2254-65 Carboxylesterase 1562 2254-66 siah binding protein 1 1563 2254-70 vacuolar ATPase, subunit M9.7 1564 2254-83 Fumarylacetoacetate hydrolase 1565 2254-84 metalloproteinase 1 1566 2254-88 alpha-spectrin 1567 2254-93 NADH dehydrogenase subunit 6 1568 2254-96 cyclophilin isoform 5 1569 2255-5 similar to mitochondrial ATPase inhibitors 1570 2255-8 yk391f12.5 1571 2255-12 Unknown 1572 2255-17 ribonucleotide reductase subunit M1 1573 2255-19 docking protein 1574 2255-22 Similar to rat 5E5 antigen 1575 2255-23 ribosomal protein S31 1576 2255-25 Similar to acyl-CoA dehydrogenase 1577 2255-28 Arginine tyrosine kinase 1578 2255-32 ribosomal protein L7a D., melanogaster 1579 2255-33 chS-Rex-s 1580 2255-39 Phosphoacetylglucosamine mutase 1581 2255-41 NADH dehydrogenase subunit 6 1582 2255-45 tRNA-glutamine synthetase 1583 2255-46 p68 1584 2255-49 ABC8 1585 2255-50 kynurenine aminotransferase 1586 2255-51 SmD homolog {Gly-Arg repeat} 1587 2255-56 epoxide hydrolase 1588 2255-60 Sec23 protein 1589 2255-62 HMG CoA synthase 1590 2255-63 dipeptidyl aminopeptidase-like protein 6 1591 2255-66 retinal rod Na+/Ca+, K+ exchanger 1592 2255-67 4-hydroxybutyrate coenzyme A transferase 1593 2255-70 hD54 + ins2 isofarm 1594 2255-73 chromaffin granule ATPase II homolog 1595 2255-77 40S ribosomal protein S10 1596 2255-79 34/67 kD laminin binding protein 1597 2255-82 RNA-binding protein lark D., melanogaster 1598 2255-86 thiol-specific antioxidant protein 1599 2256-7 Similar to Human estrogen-responsive finger protein 1600 2256-11 Trypsin 1601 2256-12 CEV14 1602 2256-16 AL021475 1603 2256-21 Heterogenous Nuclear Ribonucleoprotein C1 1604 2256-22 b4 integrin interactor 1605 2256-28 testis enhanced gene transcript protein 1606 2256-31 synaptic vesicle protein 2B 1607 2256-40 TNF-alpha stimulated ABC protein 1608 2256-42 carboxypeptidase A 1609 2256-46 pherophorin S 1610 2256-52 Fo-ATP synthase subunit b 1611 2256-54 PDGF associated protein 1612 2256-58 S20 ribosomal protein 1613 2256-64 ribosomal protein S9 1614 2256-69 elongation factor 1-gamma Artemia sp 1615 2256-70 conserved hypothetical protein 1616 2256-72 fructose 1,6 bisphosphate-aldolase 4C 1617 2256-73 troponin-T 1618 2256-80 SRP14 1619 2256-82 succinyl-CoA synthetase alpha subunit 1620 2256-89 Csa-19 1621 2256-92 Sacm21 1622 2256-94 apoptosis inhibitor Cydia pomonella granulosis virus 1623 2256-96 ribosomal protein L22
[0067] Table III represents a variety of flea HNC nucleic acid molecules of the present invention.
TABLE III SEQ ID NO: Name 567 2096-19NB.HNC 568 2096-25NB.HNC 569 2096-48NB.HNC 570 2096-50NB.HNC 571 2096-52NB.HNC 572 2096-55NB.HNC 573 2097-09NB.HNC 574 2097-15NB.HNC 575 2097-20NB.HNC 576 2097-22NB.HNC 577 2097-32NB.HNC 578 2097-45NB.HNC 579 2097-46NB.HNC 580 2097-47NB.HNC 581 2097-56NB.HNC 582 2097-64NB.HNC 583 2098-04NB.HNC 584 2098-40NB.HNC 585 2098-43NB.HNC 586 2099-9NB.HNC 587 2100-10NB.HNC 588 2100-45NB.HNC 589 2100-47NB.HNC 590 2100-56NB.HNC 591 2100-63NB.HNC 592 2110-41NB.HNC 593 2110-53NB.HNC 594 2112-12NB.HNC 595 2112-35NB.HNC 596 2113-17NB.HNC 597 2115-16NB.HNC 598 2115-22NB.HNC 599 2115-3NB.HNC 600 2116-19NB.HNC 601 2116-24NB.HNC 602 2116-27NB.HNC 603 2116-41NB.HNC 604 2116-59NB.HNC 605 2116-64NB.HNC 606 2117-05NB.HNC 607 2117-09NB.HNC 608 2117-11NB.HNC 609 2117-53NB.HNC 610 2118-03NB.HNC 611 2122-39NB.HNC 612 2123-25NB.HNC 613 2124-40NB.HNC 614 2124-62NB.HNC 615 2131-22NB.HNC 616 2131-32NB.HNC 617 2132-15NB.HNC 618 2132-28NB.HNC 619 2132-65NB.HNC 620 2132-9NB.HNC 621 2137-19NB.HNC 622 2137-24NB.HNC 623 2138-05NB.HNC 624 2138-51NB.HNC 625 2139-31NB.HNC 626 2139-41NB.HNC 627 2139-60NB.HNC 628 2140-13NB.HNC 629 2140-15NB.HNC 630 2140-18NB.HNC 631 2140-54NB.HNC 632 2141-16NB.HNC 633 2141-59NB.HNC 634 2142-16NB.HNC 635 2142-18NB.HNC 636 2143-06NB.HNC 637 2143-07NB.HNC 638 2143-33NB.HNC 639 2143-54NB.HNC 640 2168-06NB.HNC 641 2168-09NB.HNC 642 2168-42NB.HNC 643 2168-79NB.HNC 644 2168-82NB.HNC 645 2170-04NB.HNC 646 2170-08NB.HNC 647 2170-82NB.HNC 648 2172-39NB.HNC 649 2172-59NB.HNC 650 2172-60NB.HNC 651 2172-77NB.HNC 652 2174-14NB.HNC 653 2174-17NB.HNC 654 2174-41NB.HNC 655 2174-49NB.HNC 656 2174-59NB.HNC 657 2174-68NB.HNC 658 2176-21NB.HNC 659 2176-34NB.HNC 660 2176-47NB.HNC 661 2176-56NB.HNC 662 2176-62NB.HNC 663 2176-63NB.HNC 664 2176-64NB.HNC 665 2176-65NB.HNC 666 2176-75NB.HNC 667 2178-05NB.HNC 668 2178-13NB.HNC 669 2178-23NB.HNC 670 2178-25NB.HNC 671 2178-41NB.HNC 672 2178-56NB.HNC 673 2178-57NB.HNC 674 2178-58NB.HNC 675 2178-67NB.HNC 676 2178-72NB.HNC 677 2178-78NB.HNC 678 2178-80NB.HNC 679 2178-90NB.HNC 680 2178-91NB.HNC 681 2178-95NB.HNC 682 2180-05NB.HNC 683 2180-18NB.HNC 684 2180-20NB.HNC 685 2180-32NB.HNC 686 2180-59NB.HNC 687 2180-62NB.HNC 688 2180-74NB.HNC 689 2180-78NB.HNC 690 2180-79NB.HNC 691 2180-88NB.HNC 692 2180-90NB.HNC 693 2182-07NB.HNC 694 2182-12NB.HNC 695 2182-13NB.HNC 696 2182-27NB.HNC 697 2182-2NB.HNC 698 2182-46NB.HNC 699 2182-55NB.HNC 700 2182-57NB.HNC 701 2182-63NB.HNC 702 2182-64NB.HNC 703 2182-83NB.HNC 704 2182-86NB.HNC 705 2182-88NB.HNC 706 2182-90NB.HNC 707 2182-92NB.HNC 708 2182-94NB.HNC 709 2184-15NB.HNC 710 2184-37NB.HNC 711 2184-65NB.HNC 712 2186-14NB.HNC 713 2186-45NB.HNC 714 2186-50NB.HNC 715 2186-52NB.HNC 716 2186-60NB.HNC 717 2186-62NB.HNC 718 2186-63NB.HNC 719 2186-68NB.HNC 720 2186-69NB.HNC 721 2211-19NB.HNC 722 2211-23NB.HNC 723 2211-29N8.HNC 724 2211-30NB.HNC 725 2211-43NB.HNC 726 2211-52NB.HNC 727 2211-64NB.HNC 728 2212-30NB.HNC 729 2212-31NB.HNC 730 2212-71NB.HNC 731 2212-72NB.HNC 732 2212-73NB.HNC 733 2212-81NB.HNC 734 2212-85NB.HNC 735 2212-87NB.HNC 736 2212-91NB.HNC 737 2212-96NB.HNC 738 2212-9NB.HNC 739 2213-08NB.HNC 740 2213-09NB.HNC 741 2213-11NB.HNC 742 2213-12NB.HNC 743 2213-18NB.HNC 744 2213-34NB.HNC 745 2213-53NB.HNC 746 2213-58NB.HNC 747 2213-67NB.HNC 748 2213-79NB.HNC 749 2214-02NB.HNC 750 2214-03NB.HNC 751 2214-05NB.HNC 752 2214-07NB.HNC 753 2214-15NB.HNC 754 2214-23NB.HNC 755 2214-30NB.HNC 756 2214-36NB.HNC 757 2214-37NB.HNC 758 2214-40NB.HNC 759 2214-43NB.HNC 760 2214-53NB.HNC 761 2214-57NB.HNC 762 2214-60NB.HNC 763 2214-61NB.HNC 764 2214-73NB.HNC 765 2214-76NB.HNC 766 2214-80NB.HNC 767 2215-07NB.HNC 768 2215-15NB.HNC 769 2215-31NB.HNC 770 2215-41NB.HNC 771 2215-51NB.HNC 772 2215-80NB.HNC 773 2215-85NB.HNC 774 2215-91NB.HNC 775 2217-14NB.HNC 776 2217-16NB.HNC 777 2217-33NB.HNC 778 2217-39NB.HNC 779 2217-78NB.HNC 780 2217-92NB.HNC 781 2218-15NB.HNC 782 2218-19NB.HNC 783 2218-26NB.HNC 784 2218-36NB.HNC 785 2218-41NB.HNC 786 2218-56NB.HNC 787 2218-58NB.HNC 788 2218-69NB.HNC 789 2218-71NB.HNC 790 2218-76NB.HNC 791 2218-77NB.HNC 792 2218-84NB.HNC 793 2218-96NB.HNC 794 2219-11NB.HNC 795 2219-13NB.HNC 796 2219-17NB.HNC 797 2219-19NB.HNC 798 2219-20NB.HNC 799 2219-22NB.HNC 800 2219-23NB.HNC 801 2219-32NB.HNC 802 2219-45NB.HNC 803 2219-49NB.HNC 804 2219-51NB.HNC 805 2219-72NB.HNC 806 2219-80NB.HNC 807 2219-952122-39NB.HNC 2220-02NB.HNC 808 2220-02NB.HNC 809 2220-27NB.HNC 810 2220-32NB.HNC 811 2220-53NB.HNC 812 2220-60NB.HNC 813 2220-66NB.HNC 814 2221-06NB.HNC 815 2221-15NB.HNC 816 2221-18NB.HNC 817 2221-20NB.HNC 818 2221-24NB.HNC 819 2221-45NB.HNC 820 2221-46NB.HNC 821 2221-48NB.HNC 822 2221-54NB.HNC 823 2221-55NB.HNC 824 2221-59NB.HNC 825 2221-61NB.HNC 826 2221-62NB.HNC 827 2221-70NB.HNC 828 2221-86NB.HNC 829 2221-87NB.HNC 830 2221-95NB.HNC 831 2223u-18NB.HNC 832 2223u-22NB.HNC 833 2223u-23NB.HNC 834 2223u-31NB.HNC 835 2223u-33NB.HNC 836 2223u-36NB.HNC 837 2223u-67NB.HNC 838 2223u-85NB.HNC 839 2224u-05NB.HNC 840 2224u-07NB.HNC 841 2224u-10NB.HNC 842 2224u-11NB.HNC 843 2224u-15NB.HNC 844 2224u-25NB.HNC 845 2224u-27NB.HNC 846 2224u-44NB.HNC 847 2224u-52NB.HNC 848 2224u-62NB.HNC 849 2224u-70NB.HNC 850 2224u-71NB.HNC 851 2224u-79NB.HNC 852 2225u-11NB.HNC 853 2225u-20NB.HNC 854 2225u-23NB.HNC 855 2225u-28NB.HNC 856 2225u-55NB.HNC 857 2225u-59NB.HNC 858 2225u-64NB.HNC 859 2225u-77NB.HNC 860 2225u-95NB.HNC 861 2226-932122-39NB.HNC 862 2226u-07NB.HNC 863 2226u-19NB.HNC 864 2226u-39NB.HNC 865 2226u-45NB.HNC 866 2226u-49NB.HNC 867 2226u-54NB.HNC 868 2226u-71NB.HNC 869 2226u-77NB.HNC 870 2226u-83NB.HNC 871 2226u-91NB.HNC 872 2227u-12NB.HNC 873 2227u-13NB.HNC 874 2227u-23NB.HNC 875 2227u-26NB.HNC 876 2227u-30NB.HNC 877 2227u-31NB.HNC 878 2227u-33NB.HNC 879 2227u-43NB.HNC 880 2227u-51NB.HNC 881 2227u-60NB.HNC 882 2227u-93NB.HNC 883 2228u-04NB.HNC 884 2228u-09NB.HNC 885 2228u-12NB.HNC 886 2228u-21NB.HNC 887 2228u-26NB.HNC 888 2228u-49NB.HNC 889 2228u-54NB.HNC 890 2228u-55NB.HNC 891 2228u-61NB.HNC 892 2228u-65NB.HNC 893 2228u-79NB.HNC 894 2228u-90NB.HNC 1624 2222-7 1625 2222-16 1626 2222-19 1627 2222-39 1628 2222-56 1629 2222-59 1630 2222-79 1631 2222-89 1632 2228-4 1633 2228-9 1634 2228-12 1635 2228-21 1636 2228-26 1637 2228-49 1638 2228-54 1639 2228-61 1640 2228-65 1641 2228-79 1642 2228-90 1643 2245-5 1644 2245-7 1645 2245-15 1646 2245-16 1647 2245-17 1648 2245-20 1649 2245-35 1650 2245-38 1651 2245-39 1652 2245-51 1653 2245-52 1654 2245-57 1655 2246-13 1656 2246-19 1657 2246-25 1658 2246-27 1659 2246-29 1660 2246-40 1661 2246-45 1662 2246-52 1663 2246-64 1664 2246-66 1665 2246-74 1666 2246-82 1667 2247-6 1668 2247-17 1669 2247-29 1670 2247-31 1671 2247-36 1672 2247-40 1673 2247-46 1674 2247-50 1675 2247-54 1676 2247-63 1677 2247-66 1678 2247-68 1679 2247-69 1680 2247-81 1681 2247-82 1682 2247-95 1683 2248-7 1684 2248-18 1685 2248-32 1686 2248-41 1687 2248-50 1688 2248-54 1689 2248-60 1690 2248-62 1691 2248-65 1692 2248-86 1693 2248-94 1694 2249-6 1695 2249-30 1696 2249-35 1697 2249-36 1698 2249-68 1699 2249-74 1700 2249-79 1701 2250-20 1702 2250-24 1703 2251-7 1704 2251-21 1705 2251-25 1706 2251-38 1707 2251-58 1708 2252-7 1709 2252-15 1710 2252-19 1711 2252-24 1712 2252-26 1713 2252-27 1714 2252-32 1715 2252-36 1716 2252-37 1717 2252-69 1718 2252-78
[0068] Table IV represents a variety of flea HMT nucleic acid molecules of the present invention.
TABLE IV SEQ ID NO: Name SEQ ID NO: Name 895 2084-02.HMTNB 936 2086-44.HMTNB 896 2084-05.HMTNB 937 2086-54.HMTNB 897 2084-07.HMTNB 938 2086-55.HMTNB 898 2084-09.HMTNB 939 2086-58.HMTNB 899 2084-15.HMTNB 940 2087-09.HMTNB 900 2084-17.HMTNB 941 2087-17.HMTNB 901 2084-18.HMTNB 942 2087-28.HMTNB 902 2084-21.HMTNB 943 2087-33.HMTNB 903 2084-22.HMTNB 944 2087-35.HMTNB 904 2084-30.HMTNB 945 2087-51.HMTNB 905 2084-33.HMTNB 946 2087-54.HMTNB 906 2084-36.HMTNB 947 2088-07.HMTNB 907 2084-37.HMTNB 948 2088-17.HMTNB 908 2084-38.HMTNB 949 2088-35.HMTNB 909 2084-39.HMTNB 950 2088-52.HMTNB 910 2084-43.HMTNB 951 2088-59.HMTNB 911 2084-50.HMTNB 952 2089-12.HMTNB 912 2084-54.HMTNB 953 2089-14.HMTNB 913 2084-56.HMTNB 954 2089-33.HMTNB 914 2084-59.HMTNB 955 2089-36.HMTNB 915 2085-03.HMTNB 956 2089-51.HMTNB 916 2085-13.HMTNB 957 2089-60.HMTNB 917 2085-35.HMTNB 958 2090-11.HMTNB 918 2085-38.HMTNB 959 2090-27.HMTNB 919 2085-39.HMTNB 960 2090-33.HMTNB 920 2085-49.HMTNB 961 2090-44.HMTNB 921 2085-53.HMTNB 962 2090-57.HMTNB 922 2085-58.HMTNB 963 2091-11.HMTNB 923 2085-61.HMTNB 964 2091-22.HMTNB 924 2086-05.HMTNB 965 2091-23.HMTNB 925 2086-10.HMTNB 966 2091-35.HMTNB 926 2086-13.HMTNB 967 2091-63.HMTNB 927 2086-15.HMTNB 968 2092-11.HMTNB 928 2086-20.HMTNB 969 2092-16.HMTNB 929 2086-25.HMTNB 970 2092-40.HMTNB 930 2086-32.HMTNB 971 2092-42.HMTNB 931 2086-33.HMTNB 972 2092-46.HMTNB 932 2086-34.HMTNB 973 2092-60.HMTNB 933 2086-37.HMTNB 974 2093-20.HMTNB 934 2086-41.HMTNB 975 2093-23.HMTNB 935 2086-43.HMTNB 976 2093-43.HMTNB 977 2093-48.HMTNB 1025 2106-27.HMTNB 978 2093-50.HMTNB 1026 2106-29.HMTNB 979 2093-62.HMTNB 1027 2106-34.HMTNB 980 2093-63.HMTNB 1028 2106-48.HMTNB 981 2094-08.HMTNB 1029 2106-50.HMTNB 982 2094-26.HMTNB 1030 2106-64.HMTNB 983 2094-33.HMTNB 1031 2107-02.HMTNB 984 2094-47.HMTNB 1032 2107-10.HMTNB 985 2094-50.HMTNB 1033 2107-37.HMTNB 986 2094-62.HMTNB 1034 2108-03.HMTNB 987 2095-04.HMTNB 1035 2108-23.HMTNB 988 2095-10.HMTNB 1036 2108-46.HMTNB 989 2095-12.HMTNB 1037 2108-47.HMTNB 990 2095-13.HMTNB 1038 2108-48.HMTNB 991 2095-15.HMTNB 1039 2108-49.HMTNB 992 2095-20.HMTNB 1040 2108-63.HMTNB 993 2095-22.HMTNB 1041 2109-04.HMTNB 994 2095-31.HMTNB 1042 2109-06.HMTNB 995 2095-33.HMTNB 1043 2109-37.HMTNB 996 2095-34.HMTNB 1044 2109-38.HMTNB 997 2095-36.HMTNB 1045 2109-44.HMTNB 998 2095-40.HMTNB 1046 2154-08.HMTNB 999 2095-48.HMTNB 1047 2154-09.HMTNB 1000 2102-12.HMTNB 1048 2154-10.HMTNB 1001 2102-16.HMTNB 1049 2154-28.HMTNB 1002 2102-18.HMTNB 1050 2154-30.HMTNB 1003 2102-19.HMTNB 1051 2154-45.HMTNB 1004 2102-20.HMTNB 1052 2154-46.HMTNB 1005 2102-29.HMTNB 1053 2154-61.HMTNB 1006 2102-35.HMTNB 1054 2154-71.HMTNB 1007 2102-37.HMTNB 1055 2154-81.HMTNB 1008 2102-38.HMTNB 1056 2154-83.HMTNB 1009 2102-41.HMTNB 1057 2156-02.HMTNB 1010 2102-47.HMTNB 1058 2156-06.HMTNB 1011 2103-02.HMTNB 1059 2156-18.HMTNB 1012 2103-09.HMTNB 1060 2156-27.HMTNB 1013 2103-45.HMTNB 1061 2156-43.HMTNB 1014 2103-56.HMTNB 1062 2156-48.HMTNB 1015 2103-58.HMTNB 1063 2156-50.HMTNB 1016 2104-58.HMTNB 1064 2157-16.HMTNB 1017 2104-60.HMTNB 1065 2157-34.HMTNB 1018 2104-61.HMTNB 1066 2157-45.HMTNB 1019 2105-02.HMTNB 1067 2157-70.HMTNB 1020 2105-20.HMTNB 1068 2157-75.HMTNB 1021 2105-35.HMTNB 1069 2157-79.HMTNB 1022 2105-42.HMTNB 1070 2157-86.HMTNB 1023 2105-44.HMTNB 1071 2158-02.HMTNB 1024 2106-05.HMTNB 1072 2158-14.HMTNB 1073 2158-19.HMTNB 1121 2163-11.HMTNB 1074 2158-22.HMTNB 1122 2163-18.HMTNB 1075 2158-27.HMTNB 1123 2163-23.HMTNB 1076 2158-34.HMTNB 1124 2163-25.HMTNB 1077 2158-37.HMTNB 1125 2163-43.HMTNB 1078 2158-39.HMTNB 1126 2163-50.HMTNB 1079 2159-07.HMTNB 1127 2163-61.HMTNB 1080 2159-09.HMTNB 1128 2163-65.HMTNB 1081 2159-17.HMTNB 1129 2163-73.HMTNB 1082 2159-34.HMTNB 1130 2163-77.HMTNB 1083 2159-35.HMTNB 1131 2163-87.HMTNB 1084 2159-60.HMTNB 1132 2163-93.HMTNB 1085 2160-16.HMTNB 1133 2163-95.HMTNB 1086 2160-17.HMTNB 1134 2165-04.HMTNB 1087 2160-29.HMTNB 1135 2165-06.HMTNB 1088 2160-30.HMTNB 1136 2165-24.HMTNB 1089 2160-32.HMTNB 1137 2165-45.HMTNB 1090 2160-39.HMTNB 1138 2165-59.HMTNB 1091 2160-49.HMTNB 1139 2165-65.HMTNB 1092 2160-53.HMTNB 1140 2166-02.HMTNB 1093 2160-54.HMTNB 1141 2166-12.HMTNB 1094 2160-55.HMTNB 1142 2166-42.HMTNB 1095 2160-77.HMTNB 1143 2166-46.HMTNB 1096 2160-82.HMTNB 1144 2166-47.HMTNB 1097 2160-89.HMTNB 1145 2167-07.HMTNB 1098 2160-91.HMTNB 1146 2167-16.HMTNB 1099 2161-13.HMTNB 1147 2167-42.HMTNB 1100 2161-19.HMTNB 1148 2167-65.HMTNB 1101 2161-45.HMTNB 1149 2167-66.HMTNB 1102 2161-57.HMTNB 1150 2167-79.HMTNB 1103 2161-60.HMTNB 1151 2167-90.HMTNB 1104 2161-79.HMTNB 1152 2167-94.HMTNB 1105 2161-83.HMTNB 1153 2169-05.HMTNB 1106 2161-90.HMTNB 1154 2169-12.HMTNB 1107 2161-94.HMTNB 1155 2169-16.HMTNB 1108 2162-05.HMTNB 1156 2169-17.HMTNB 1109 2162-12.HMTNB 1157 2169-19.HMTNB 1110 2162-13.HMTNB 1158 2169-22.HMTNB 1111 2162-18.HMTNB 1159 2169-26.HMTNB 1112 2162-35.HMTNB 1160 2169-33.HMTNB 1113 2162-41.HMTNB 1161 2169-42.HMTNB 1114 2162-50.HMTNB 1162 2169-46.HMTNB 1115 2162-59.HMTNB 1163 2169-47.HMTNB 1116 2162-63.HMTNB 1164 2169-57.HMTNB 1117 2162-71.HMTNB 1165 2169-69.HMTNB 1118 2162-75.HMTNB 1166 2171-06.HMTNB 1119 2162-78.HMTNB 1167 2171-09.HMTNB 1120 2163-07.HMTNB 1168 2171-11.HMTNB 1169 2171-29.HMTNB 1217 2183-70.HMTNB 1170 2171-33.HMTNB 1218 2185-05.HMTNB 1171 2171-35.HMTNB 1219 2185-10.HMTNB 1172 2171-41.HMTNB 1220 2185-12.HMTNB 1173 2171-54.HMTNB 1221 2185-18.HMTNB 1174 2171-57.HMTNB 1222 2185-43.HMTNB 1175 2171-69.HMTNB 1223 2185-49.HMTNB 1176 2171-82.HMTNB 1224 2185-54.HMTNB 1177 2171-84.HMTNB 1225 2185-82.HMTNB 1178 2171-85.HMTNB 1226 2187-21.HMTNB 1179 2173-12.HMTNB 1227 2187-37.HMTNB 1180 2173-34.HMTNB 1228 2187-47.HMTNB 1181 2173-42.HMTNB 1229 2187-93.HMTNB 1182 2173-48.HMTNB 1230 2188-22.HMTNB 1183 2173-54.HMTNB 1231 2188-29.HMTNB 1184 2173-57.HMTNB 1232 2188-32.HMTNB 1185 2173-75.HMTNB 1233 2188-52.HMTNB 1186 2173-86.HMTNB 1234 2188-54.HMTNB 1187 2173-91.HMTNB 1235 2188-72.HMTNB 1188 2175-06.HMTNB 1236 2188-92.HMTNB 1189 2175-15.HMTNB 1237 2189-31.HMTNB 1190 2175-20.HMTNB 1238 2189-56.HMTNB 1191 2175-58.HMTNB 1239 2189-75.HMTNB 1192 2175-96.HMTNB 1240 2189-84.HMTNB 1193 2177-16.HMTNB 1241 2191-23.HMTNB 1194 2177-70.HMTNB 1242 2191-38.HMTNB 1195 2177-86.HMTNB 1243 2191-58.HMTNB 1196 2179-02.HMTNB 1244 2191-73.HMTNB 1197 2179-03.HMTNB 1245 2191-77.HMTNB 1198 2179-19.HMTNB 1246 2191-90.HMTNB 1199 2179-22.HMTNB 1247 2191-94.HMTNB 1200 2179-29.HMTNB 1248 2191-96.HMTNB 1201 2179-39.HMTNB 1249 2192-03.HMTNB 1202 2179-63.HMTNB 1250 2192-14.HMTNB 1203 2181-04.HMTNB 1251 2192-36.HMTNB 1204 2181-24.HMTNB 1252 2192-46.HMTNB 1205 2181-35.HMTNB 1253 2192-88.HMTNB 1206 2181-66.HMTNB 1254 2194-07.HMTNB 1207 2181-75.HMTNB 1255 2194-13.HMTNB 1208 2181-76.HMTNB 1256 2194-16.HMTNB 1209 2181-84.HMTNB 1257 2194-18.HMTNB 1210 2183-05.HMTNB 1258 2194-28.HMTNB 1211 2183-13.HMTNB 1259 2195-06.HMTNB 1212 2183-17.HMTNB 1260 2195-47.HMTNB 1213 2183-28.HMTNB 1261 2195-60.HMTNB 1214 2183-45.HMTNB 1262 2196-18.HMTNB 1215 2183-50.HMTNB 1263 2196-30.HMTNB 1216 2183-51.HMTNB 1264 2196-53.HMTNB 1265 2196-65.HMTNB 1313 2231-44u.HMTNB 1266 2196-76.HMTNB 1314 2231-50u.HMTNB 1267 2197-28.HMTNB 1315 2231-51u.HMTNB 1268 2197-46.HMTNB 1316 2231-63u.HMTNB 1269 2197-51.HMTNB 1317 2231-68u.HMTNB 1270 2197-59.HMTNB 1318 2231-74u.HMTNB 1271 2202-96.HMTNB 1319 2231-82u.HMTNB 1272 2203-36.HMTNB 1320 2231-85u.HMTNB 1273 2204-09.HMTNB 1321 2231-88u.HMTNB 1274 2205-11.HMTNB 1322 2231-94u.HMTNB 1275 2205-33.HMTNB 1323 2231-95u.HMTNB 1276 2205-43.HMTNB 1324 2232-03u.HMTNB 1277 2205-85.HMTNB 1325 2232-11u.HMTNB 1278 2229-08u.HMTNB 1326 2232-19u.HMTNB 1279 2229-10u.HMTNB 1327 2232-25u.HMTNB 1280 2229-12u.HMTNB 1328 2232-30u.HMTNB 1281 2229-14u.HMTNB 1329 2232-44u.HMTNB 1282 2229-27u.HMTNB 1330 2232-50u.HMTNB 1283 2229-40u.HMTNB 1331 2232-56u.HMTNB 1284 2229-45u.HMTNB 1332 2232-60u.HMTNB 1285 2229-48u.HMTNB 1333 2232-64u.HMTNB 1286 2229-50u.HMTNB 1334 2232-71u.HMTNB 1287 2229-54u.HMTNB 1335 2232-73u.HMTNB 1288 2229-56u.HMTNB 1336 2232-80u.HMTNB 1289 2229-57u.HMTNB 1337 2232-83u.HMTNB 1290 2229-59u.HMTNB 1338 2233-02u.HMTNB 1291 2229-70u.HMTNB 1339 2233-53u.HMTNB 1292 2229-87u.HMTNB 1340 2233-57u.HMTNB 1293 2229-91u.HMTNB 1341 2233-58u.HMTNB 1294 2229-95u.HMTNB 1342 2233-80u.HMTNB 1295 2230-07u.HMTNB 1343 2233-81u.HMTNB 1296 2230-11u.HMTNB 1344 2233-83u.HMTNB 1297 2230-19u.HMTNB 1345 2234-02u.HMTNB 1298 2230-27u.HMTNB 1346 2234-03u.HMTNB 1299 2230-33u.HMTNB 1347 2234-05u.HMTNB 1300 2230-41u.HMTNB 1348 2234-06u.HMTNB 1301 2230-51u.HMTNB 1349 2234-09u.HMTNB 1302 2230-56u.HMTNB 1350 2234-12u.HMTNB 1303 2230-66u.HMTNB 1351 2234-23u.HMTNB 1304 2230-71u.HMTNB 1352 2234-26u.HMTNB 1305 2230-75.HMTNB 1353 2234-46u.HMTNB 1306 2230-81u.HMTNB 1354 2234-66u.HMTNB 1307 2230-84u.HMTNB 1355 2234-67u.HMTNB 1308 2230-93u.HMTNB 1356 2234-70u.HMTNB 1309 2231-23u.HMTNB 1357 2234-74u.HMTNB 1310 2231-26u.HMTNB 1358 2234-77u.HMTNB 1311 2231-32u.HMTNB 1359 2234-82u.HMTNB 1312 2231-37u.HMTNB 1360 2234-88u.HMTNB 1361 2234-89u.HMTNB 1409 2244-47 1362 2234-90u.HMTNB 1410 2244-52 1363 2234-93u.HMTNB 1411 2244-57 1364 2240-39 1412 2244-63 1365 2240-40 1413 2244-68 1366 2240-49 1414 2244-77 1367 2240-51 1415 2244-80 1368 2240-57 1369 2240-61 1370 2240-62 1371 2241-2 1372 2241-3 1373 2241-8 1374 2241-9 1375 2241-13 1376 2241-21 1377 2241-29 1378 2241-38 1379 2241-45 1380 2241-49 1381 2241-51 1382 2241-57 1383 2241-63 1384 2241-68 1385 2241-89 1386 2241-91 1387 2243-2 1388 2243-3 1389 2243-12 1390 2243-14 1391 2243-19 1392 2243-24 1393 2243-25 1394 2243-33 1395 2243-49 1396 2243-50 1397 2243-51 1398 2243-59 1399 2243-63 1400 2243-69 1401 2243-74 1402 2243-75 1403 2243-77 1404 2244-19 1405 2244-26 1406 2244-35 1407 2244-38 1408 2244-40
[0069] In one embodiment, a gene or other nucleic acid molecule of the present invention can be an allelic variant that includes a similar but not identical sequence to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:1859, SEQ ID NO:1860, SEQ ID NO:1861, SEQ ID NO:1863, SEQ ID NO:1864, SEQ ID NO:1866, SEQ ID NO:1867, SEQ ID NO:1869, SEQ ID NO:1870, SEQ ID NO:1871, SEQ ID NO:1872, SEQ ID NO:1874, SEQ ID NO:1875, SEQ ID NO:1876, SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1880, SEQ ID NO:1881, SEQ ID NO:1882, SEQ ID NO:1884, SEQ ID NO:1885, SEQ ID NO:1886, SEQ ID NO:1887, SEQ ID NO:1889, SEQ ID NO:1890, SEQ ID NO:1891, SEQ ID NO:1892, SEQ ID NO:1893, SEQ ID NO:1894, SEQ ID NO:1895, SEQ ID NO:1896, SEQ ID NO:1898, SEQ ID NO:1899, SEQ ID NO:1900, SEQ ID NO:1901, SEQ ID NO:1903, SEQ ID NO:1904, SEQ ID NO:1905, SEQ ID NO:1906, SEQ ID NO:1907, SEQ ID NO:1908, SEQ ID NO:1909, SEQ ID NO:1910, SEQ ID NO:1911, SEQ ID NO:1912, SEQ ID NO:1913, SEQ ID NO:1914, SEQ ID NO:1916, SEQ ID NO:1917, SEQ ID NO:1918, SEQ ID NO:1919, SEQ ID NO:1921, SEQ ID NO:1922, SEQ ID NO:1923, SEQ ID NO:1924, SEQ ID NO:1926, SEQ ID NO:1927, SEQ ID NO:1928, SEQ ID NO:1929, and/or SEQ ID NO:1931 or a
[0070] In one embodiment of the present invention, isolated HMT and HNC proteins are encoded by nucleic acid molecules that hybridize under stringent hybridization conditions to genes or other nucleic acid molecules encoding flea HMT and HNC proteins, respectively. The minimal size of HMT and HNC proteins of the present invention is a size sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid (i.e., hybridizing under stringent hybridization conditions) with the complementary sequence of a nucleic acid molecule encoding the corresponding natural protein. The size of a nucleic acid molecule encoding such a protein is dependent on the nucleic acid composition and the percent homology between the flea HMT or HNC nucleic acid molecule and the complementary nucleic acid sequence. It can easily be understood that the extent of homology required to form a stable hybrid under stringent conditions can vary depending on whether the homologous sequences are interspersed throughout a given nucleic acid molecule or are clustered (i.e., localized) in distinct regions on a given nucleic acid molecule.
[0071] The minimal size of a nucleic acid molecule capable of forming a stable hybrid with a gene encoding a flea HMT or HNC protein is typically at least about 12 to about 15 nucleotides in length if the nucleic acid molecule is GC-rich and at least about 15 to about 17 bases in length if it is AT-rich. The minimal size of a nucleic acid molecule used to encode an HMT or HNC protein homologue of the present invention is from about 12 to about 18 nucleotides in length. Thus, the minimal size of HMT or HNC protein homologues of the present invention is from about 4 to about 6 amino acids in length. There is no limit, other than a practical limit, on the maximal size of a nucleic acid molecule encoding a flea HMT or HNC protein of the present invention because a nucleic acid molecule of the present invention can include a portion of a gene, an entire gene, or multiple genes. The preferred size of a protein encoded by a nucleic acid molecule of the present invention depends on whether a full-length, fusion, multivalent, or functional portion of such a protein is desired.
[0072] Stringent hybridization conditions are determined based on defined physical properties of the gene to which the nucleic acid molecule is being hybridized, and can be defined mathematically. Stringent hybridization conditions are those experimental parameters that allow an individual skilled in the art to identify significant similarities between heterologous nucleic acid molecules. These conditions are well known to those skilled in the art. See, for example, Sambrook, et al., 1989,
[0073] For nucleic acid molecules smaller than about 50 nucleotides, hybrid stability is defined by the dissociation temperature (T
[0074] A temperature of 5° C. below T
[0075] Also well known to those skilled in the art is how base pair mismatch, i.e. differences between two nucleic acid molecules being compared, including non-complementarity of bases at a given location, and gaps due to insertion or deletion of one or more bases at a given location on either of the nucleic acid molecules being compared, will affect Tm or Td for nucleic acid molecules of different sizes. For example, T
[0076] Hybridization reactions are often carried out by attaching the nucleic acid molecule to be hybridized to a solid support such as a membrane, and then hybridizing with a labeled nucleic acid molecule, typically referred to as a probe, suspended in a hybridization solution. Examples of common hybridization reaction techniques include, but are not limited to, the well-known Southern and northern blotting procedures. Typically, the actual hybridization reaction is done under non-stringent conditions, i.e., at a lower temperature and/or a higher salt concentration, and then high stringency is achieved by washing the membrane in a solution with a higher temperature and/or lower salt concentration in order to achieve the desired stringency.
[0077] For example, if the skilled artisan wished to identify a nucleic acid molecule that hybridizes under conditions that would allow less than or equal to 30% pair mismatch with a flea nucleic acid molecule of about 150 bp in length or greater, the following conditions could preferably be used. The average G+C content of flea DNA is about 37%, as calculated from known flea nucleic acid sequences. The unknown nucleic acid molecules would be attached to a support membrane, and the 150 bp probe would be labeled, e.g. with a radioactive tag. The hybridization reaction could be carried out in a solution comprising 2× SSC and 0% formamide, at a temperature of about 37° C. (low stringency conditions). Solutions of differing concentrations of SSC can be made by one of skill in the art by diluting a stock solution of 20× SSC (175.3 gram NaCl and about 88.2 gram sodium citrate in 1 liter of water, pH 7) to obtain the desired concentration of SSC. The skilled artisan would calculate the washing conditions required to allow up to 30% base pair mismatch. For example, in a wash solution comprising 1× SSC and 0% formamide, the T
[0078] Thus, to achieve hybridization with nucleic acid molecules having about 30% base pair mismatch, hybridization washes would be carried out at a temperature of less than or equal to 47.5° C. It is thus within the skill of one in the art to calculate additional hybridization temperatures based on the desired percentage base pair mismatch, formulae and G/C content disclosed herein. For example, it is appreciated by one skilled in the art that as the nucleic acid molecule to be tested for hybridization against nucleic acid molecules of the present invention having sequences specified herein becomes longer than 150 nucleotides, the T
[0079] Furthermore, it is known in the art that there are commercially available computer programs for determining the degree of similarity between two nucleic acid sequences. These computer programs include various known methods to determine the percentage identity and the number and length of gaps between hybrid nucleic acid molecules. Preferred methods to determine the percent identity among amino acid sequences and also among nucleic acid sequences include analysis using one or more of the commercially available computer programs designed to compare and analyze nucleic acid or amino acid sequences. These computer programs include, but are not limited to, the Wisconsin Package Version 9.0 sequence analysis software, available from Genetics Computer Group (GCG™), Madison, Wis., DNAsis™, available from Hitachi Software, San Bruno, Calif., and MacVector™, available from the Eastman Kodak Company, New Haven, Conn. A preferred method to determine percent identity among amino acid sequences and also among nucleic acid sequences includes using the GAP program with pair-wise comparisons within the GCGTM Wisconsin Package Version 9.0 sequence analysis software, hereinafter referred to as default parameters.
[0080] One embodiment of the present invention includes flea ALN, CBP, NKAB, LGIC, ANON, MALV, OS-D, NMDA, CLBP, NAH, CLIC, PL2, PL3, PL4, SVP, VGCC, AUP, and 7B2 proteins. A preferred flea ALN protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:6.
[0081] A preferred flea CBP protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:12.
[0082] A preferred flea NKAB protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:15 and SEQ ID NO:18.
[0083] A preferred flea LGIC protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:21 and SEQ ID NO:24, SEQ ID NO:1860, SEQ ID NO:1863, and SEQ ID NO:1866.
[0084] A preferred flea ANON protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:27 and SEQ ID NO:30.
[0085] A preferred flea MALV protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:33 and SEQ ID NO:36.
[0086] A preferred flea OS-D protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:39 and SEQ ID NO:42.
[0087] A preferred flea NMDA protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:45 and SEQ ID NO:48.
[0088] A preferred flea CLBP protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:161, SEQ ID NO:164, SEQ ID NO:167 and SEQ ID NO:170.
[0089] A preferred flea NAH protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:1869 and SEQ ID NO:1871.
[0090] A preferred flea CLIC protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:1874 and SEQ ID NO:1876.
[0091] A preferred flea PL2 protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:1879, SEQ ID NO:1881, SEQ ID NO:1884, and SEQ ID NO:1886.
[0092] A preferred flea CPL3 protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:1889 and SEQ ID NO:1891.
[0093] A preferred flea PL4 protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:1893, SEQ ID NO:1895, SEQ ID NO:1898, and SEQ ID NO:1900.
[0094] A preferred flea SVP protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:1903 and SEQ ID NO:1905.
[0095] A preferred flea VGCC protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:1907, SEQ ID NO:1909, SEQ ID NO:1911, SEQ ID NO:1913, SEQ ID NO:1916, and SEQ ID NO:1918.
[0096] A preferred flea AUP protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:1921 and SEQ ID NO:1923.
[0097] A preferred flea 7B2 protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:1926, SEQ ID NO:1928, and SEQ ID NO:1931.
[0098] A preferred flea HMT and/or HNC protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of a nucleic acid sequence complementary to a nucleic acid sequence of Table I, Table II, Table III and/or Table IV.
[0099] Another embodiment of the present invention includes a flea ALN protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising 1×SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:3 and SEQ ID NO:6.
[0100] Another embodiment of the present invention includes a flea CBP protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:9 and SEQ ID NO:13.
[0101] Another embodiment of the present invention includes a flea NKAB protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:15 and SEQ ID NO:18.
[0102] Another embodiment of the present invention includes a flea LGIC protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising IX SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:1860, SEQ ID NO:1863, and SEQ ID NO:1866.
[0103] Another embodiment of the present invention includes a flea ANON protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:27 and SEQ ID NO:30.
[0104] Another embodiment of the present invention includes a flea MALV protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:33 and SEQ ID NO:36.
[0105] Another embodiment of the present invention includes a flea OS-D protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising IX SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:39 and SEQ ID NO:42.
[0106] Another embodiment of the present invention includes a flea NMDA protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:45 and SEQ ID NO:48.
[0107] Another embodiment of the present invention includes a flea CLBP protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:155, SEQ ID NO:158, SEQ ID NO:161, SEQ ID NO:164, SEQ ID NO:167 and SEQ ID NO:170.
[0108] Another embodiment of the present invention includes a flea NAH protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:1869 and SEQ ID NO:1871.
[0109] Another embodiment of the present invention includes a flea CLIC protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:1874 and SEQ ID NO:1876.
[0110] Another embodiment of the present invention includes a flea PL2 protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:1879, SEQ ID NO:1881, SEQ IID NO:1884 and SEQ ID NO:1886.
[0111] Another embodiment of the present invention includes a flea PL3 protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:1889 and SEQ ID NO:1891.
[0112] Another embodiment of the present invention includes a flea PL4 protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:1893, SEQ ID NO:1895, SEQ ID NO:1898, and SEQ ID NO:1900.
[0113] Another embodiment of the present invention includes a flea SVP protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:1903 and SEQ ID NO:1905.
[0114] Another embodiment of the present invention includes a flea VGCC protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:1907, SEQ ID NO:1909, SEQ ID NO:1911, SEQ ID NO:1913, SEQ ID NO:1916, and SEQ ID NO:1918.
[0115] Another embodiment of the present invention includes a flea AUP protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:1921 and SEQ ID NO:1923.
[0116] Another embodiment of the present invention includes a flea 7B2 protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:1926, SEQ ID NO:1928, and SEQ ID NO:1931.
[0117] Another embodiment of the present invention includes a flea HMT and/or HNC protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, (a) hybridizing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 37° C. and (b) washing in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of a nucleic acid sequence complementary to a nucleic acid sequence of Table I, Table II, Table III and/or Table IV.
[0118] Another preferred flea ALN protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably about at least 85% identical, more preferably about at least 90% identical, and even more preferably about at least 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:1 and/or SEQ ID NO:4; also preferred are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least about 18 nucleotides. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
[0119] Another preferred flea CBP protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably at least about 85% identical, more preferably at least about 90% identical, and even more preferably at least about 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:7 and/or SEQ ID NO:10; also preferred are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least about 18 nucleotides. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
[0120] Another preferred flea NKAB protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably at least about 85% identical, more preferably at least about 90% identical, and even more preferably at least about 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:13 and/or SEQ ID NO:16; also preferred are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least about 18 nucleotides. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
[0121] Another preferred flea LGIC protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably at least about 85% identical, more preferably at least about 90% identical, and even more preferably at least about 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:1861, and/or SEQ ID NO:1864; also preferred are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least about 18 nucleotides. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
[0122] Another preferred flea ANON protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably at least about 85% identical, more preferably at least about 90% identical, and even more preferably at least about 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:25 and/or SEQ ID NO:28; also preferred are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least about 18 nucleotides. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
[0123] Another preferred flea MALV protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably at least about 85% identical, more preferably at least about 90% identical, and even more preferably at least about 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:31 and/or SEQ ID NO:34; also preferred are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least about 18 nucleotides. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
[0124] Another preferred flea OS-D protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably at least about 85% identical, more preferably at least about 90% identical, and even more preferably at least about 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:37 and/or SEQ ID NO:40; also preferred are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least about 18 nucleotides. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
[0125] Another preferred flea NMDA protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably at least about 85% identical, more preferably at least about 90% identical, and even more preferably at least about 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:43 and/or SEQ ID NO:46; also preferred are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least about 18 nucleotides. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
[0126] Another preferred flea CLBP protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably at least about 85% identical, more preferably at least about 90% identical, and even more preferably at least about 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:153, SEQ ID NO:156, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:165 and/or SEQ ID NO:168; also preferred are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least about 18 nucleotides. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
[0127] Another preferred flea NAH protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably about at least 85% identical, more preferably about at least 90% identical, and even more preferably about at least 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:1867 and/or SEQ ID NO:1870; also preferred are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least about 18 nucleotides. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
[0128] Another preferred flea CLIC protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably about at least 85% identical, more preferably about at least 90% identical, and even more preferably about at least 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:1872 and/or SEQ ID NO:1875; also preferred are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least about 18 nucleotides. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
[0129] Another preferred flea PL2 protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably about at least 85% identical, more preferably about at least 90% identical, and even more preferably about at least 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1880, SEQ ID NO:1882, and/or SEQ ID NO:1885; also preferred are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least about 18 nucleotides. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
[0130] Another preferred flea PL3 protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably about at least 85% identical, more preferably about at least 90% identical, and even more preferably about at least 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:1887 and/or SEQ ID NO:1890; also preferred are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least about 18 nucleotides. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
[0131] Another preferred flea PIA protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably about at least 85% identical, more preferably about at least 90% identical, and even more preferably about at least 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:1892, SEQ ID NO:1894, SEQ ID NO:1896 and/or SEQ ID NO:1899; also preferred are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least about 18 nucleotides. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
[0132] Another preferred flea SVP protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably about at least 85% identical, more preferably about at least 90% identical, and even more preferably about at least 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:1901 and/or SEQ ID NO:1904; also preferred are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least about 18 nucleotides. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
[0133] Another preferred flea VGCC protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably about at least 85% identical, more preferably about at least 90% identical, and even more preferably about at least 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:1906, SEQ ID NO:1908, SEQ ID NO:1910, SEQ ID NO:1912, SEQ ID NO:1914 and/or SEQ ID NO:1917; also preferred are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least about 18 nucleotides. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
[0134] Another preferred flea AUP protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably about at least 85% identical, more preferably about at least 90% identical, and even more preferably about at least 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:1919 and/or SEQ ID NO:1922; also preferred are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least about 18 nucleotides. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
[0135] Another preferred flea 7B2 protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably about at least 85% identical, more preferably about at least 90% identical, and even more preferably about at least 95% identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO:1924, SEQ ID NO:1927 and/or SEQ ID NO:1929; also preferred are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least about 18 nucleotides. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
[0136] Another preferred flea HMT and/or HNC protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least about 70% identical, more preferably at least about 75% identical, more preferably at least about 80% identical, more preferably at least about 85% identical, more preferably at least about 90% identical, and even more preferably at least about 95% identical to a nucleic acid molecule having a nucleic acid sequence of Table I, Table II, Table III and/or Table IV; also preferred are fragments (i.e. portions) of such proteins encoded by nucleic acid molecules that are at least about 18 nucleotides. Percent identity as used herein is determined using the Compare function by maximum matching within the program DNAsis Version 2.1 using default parameters.
[0137] Additional preferred flea ALN proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:2 or SEQ ID NO:5, and proteins comprising homologues of a protein having the amino acid sequence SEQ ID NO:2 or SEQ ID NO:5, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ID NO:2 or SEQ ID NO:5. Likewise, also preferred are proteins encoded by nucleic acid molecules comprising nucleic acid sequence SEQ ID NO:1 and/or SEQ ID NO:4, or by homologues thereof.
[0138] Additional preferred flea CBP proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:8 or SEQ ID NO:11, and proteins comprising homologues of a protein having the amino acid sequence SEQ ID NO:8 or SEQ ID NO:11, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ID NO:8 or SEQ ID NO:11. Likewise, also preferred are proteins encoded by nucleic acid molecules comprising nucleic acid sequence SEQ ID NO:7 and/or SEQ ID NO:10, or by homologues thereof Additional preferred flea NKAB proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:14 or SEQ ID NO:17, and proteins comprising homologues of a protein having the amino acid sequence SEQ ID NO:14 or SEQ ID NO:17, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ID NO:14 or SEQ ID NO:17. Likewise, also preferred are proteins encoded by nucleic acid molecules comprising nucleic acid sequence SEQ ID NO:13 and/or SEQ ID NO:16, or by homologues thereof Additional preferred flea LGIC proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:1862, and proteins comprising homologues of a protein having the amino acid sequence SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:1862, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ID NO:20, SEQ ID NO:23 or SEQ ID NO:1862. Likewise, also preferred are proteins encoded by nucleic acid molecules comprising nucleic acid sequence SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:1859, SEQ ID NO:1861 and/or SEQ ID NO:1864 or by homologues thereof.
[0139] Additional preferred flea ANON proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:26 or SEQ ID NO:29, and proteins comprising homologues of a protein having the amino acid sequence SEQ ID NO:26 or SEQ ID NO:29, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ID NO:26 or SEQ ID NO:29. Likewise, also preferred are proteins encoded by nucleic acid molecules comprising nucleic acid sequence SEQ ID NO:25 and/or SEQ ID NO:28, or by homologues thereof.
[0140] Additional preferred flea MALV proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:32 or SEQ ID NO:35, and proteins comprising homologues of a protein having the amino acid sequence SEQ ID NO:32 or SEQ ID NO:35, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ID NO:32 or SEQ ID NO:35. Likewise, also preferred are proteins encoded by nucleic acid molecules comprising nucleic acid sequence SEQ ID NO:31 and/or SEQ ID NO:34, or by homologues thereof.
[0141] Additional preferred flea OS-D proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:38 or SEQ ID NO:41, and proteins comprising homologues of a protein having the amino acid sequence SEQ ID NO:38 or SEQ ID NO:41, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ID NO:38 or SEQ ID NO:41. Likewise, also preferred are proteins encoded by nucleic acid molecules comprising nucleic acid sequence SEQ ID NO:37 and/or SEQ ID NO:40, or by homologues thereof.
[0142] Additional preferred flea NMDA proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:44 or SEQ ID NO:47, and proteins comprising homologues of a protein having the amino acid sequence SEQ ID NO:44 or SEQ ID NO:47, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ID NO:44 or SEQ ID NO:47. Likewise, also preferred are proteins encoded by nucleic acid molecules comprising nucleic acid sequence SEQ ID NO:43 and/or SEQ ID NO:46, or by homologues thereof.
[0143] Additional preferred flea CLBP proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:154, SEQ ID NO:157, SEQ ID NO:160, SEQ ID NO:163, SEQ ID NO:166 or SEQ ID NO:169, and proteins comprising homologues of a protein having the amino acid sequence SEQ ID NO:154, SEQ ID NO:157, SEQ ID NO:160, SEQ ID NO:163, SEQ ID NO:166 or SEQ ID NO:169, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ID NO:154, SEQ ID NO:157, SEQ ID NO:160, SEQ ID NO:163, SEQ ID NO:166 or SEQ ID NO:169. Likewise, also preferred are proteins encoded by nucleic acid molecules comprising nucleic acid sequence SEQ ID NO:153, SEQ ID NO:156, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:165 and/or SEQ ID NO:168, or by homologues thereof.
[0144] Additional preferred flea NAH proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:1868, and proteins comprising homologues of a protein having the amino acid sequence SEQ ID NO:1868, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ID NO:1868. Likewise, also preferred are proteins encoded by nucleic acid molecules comprising nucleic acid sequence SEQ ID NO:1867 and/or SEQ ID NO:1870, or by homologues thereof.
[0145] Additional preferred flea CLIC proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:1873, and proteins comprising homologues of a protein having the amino acid sequence SEQ ID NO:1873, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ID NO:1873. Likewise, also preferred are proteins encoded by nucleic acid molecules comprising nucleic acid sequence SEQ ID NO:1872 and/or SEQ ID NO:1875, or by homologues thereof.
[0146] Additional preferred flea PL2 proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:1883, and proteins comprising homologues of a protein having the amino acid sequence SEQ ID NO:1883, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ID NO:1883. Likewise, also preferred are proteins encoded by nucleic acid molecules comprising nucleic acid sequence SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1880, SEQ ID NO:1882 and/or SEQ ID NO:1885, or by homologues thereof.
[0147] Additional preferred flea PL3 proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:1888, and proteins comprising homologues of a protein having the amino acid sequence SEQ ID NO:1888, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ID NO:1888. Likewise, also preferred are proteins encoded by nucleic acid molecules comprising nucleic acid sequence SEQ ID NO:1887 and/or SEQ ID NO:1890, or by homologues thereof.
[0148] Additional preferred flea PL4 proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:1897, and proteins comprising homologues of a protein having the amino acid sequence SEQ ID NO:1897, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ID NO:1897. Likewise, also preferred are proteins encoded by nucleic acid molecules comprising nucleic acid sequence SEQ ID NO:1892, SEQ ID NO:1894, SEQ ID NO:1896 and/or SEQ ID NO:1899, or by homologues thereof.
[0149] Additional preferred flea SVP proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:1902, and proteins comprising homologues of a protein having the amino acid sequence SEQ ID NO:1902, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ID NO:1902. Likewise, also preferred are proteins encoded by nucleic acid molecules comprising nucleic acid sequence SEQ ID NO:1901 and/or SEQ ID NO:1904, or by homologues thereof.
[0150] Additional preferred flea VGCC proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:1915, and proteins comprising homologues of a protein having the amino acid sequence SEQ ID NO:1915, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ID NO:1915. Likewise, also preferred are proteins encoded by nucleic acid molecules comprising nucleic acid sequence SEQ ID NO:1906, SEQ ID NO:1908, SEQ ID NO:1910, SEQ ID NO:1912, SEQ ID NO:1914 and/or SEQ ID NO:1917, or by homologues thereof.
[0151] Additional preferred flea AUP proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:1920, and proteins comprising homologues of a protein having the amino acid sequence SEQ ID NO:1920, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ID NO:1920. Likewise, also preferred are proteins encoded by nucleic acid molecules comprising nucleic acid sequence SEQ ID NO:1919 and/or SEQ ID NO:1922, or by homologues thereof.
[0152] Additional preferred flea 7B2 proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:1925 or SEQ ID NO:1930, and proteins comprising homologues of a protein having the amino acid sequence SEQ ID NO:1925 or SEQ ID NO:1930, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ID NO:1925 or SEQ ID NO:1930. Likewise, also preferred are proteins encoded by nucleic acid molecules comprising nucleic acid sequence SEQ ID NO:1924, SEQ ID NO:1927 and/or SEQ ID NO:1929, or by homologues thereof.
[0153] Additional preferred flea HMT and/or HNC proteins of the present invention include proteins having an amino acid sequence encoded by a nucleic acid sequence of Table I, Table II, Table III and/or Table IV, and proteins comprising homologues of a protein encoded by a nucleic acid sequence of Table I, Table II, Table III and/or Table IV, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein encoded by a nucleic acid sequence of Table I, Table II, Table III and/or Table IV.
[0154] A preferred isolated protein of the present invention is a protein encoded by at least one of the following nucleic acid molecules: nCfALN
[0155] Preferred proteins of the present invention include proteins that are at least about 70%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, even more preferably at least about 95%, and even more preferably about 100% identical to PCfALN
[0156] Other preferred HMT and HNC proteins of the present invention include proteins having amino acid sequences that are at least about 70%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, even more preferably at least about 95%, and even more preferably about 100% identical to amino acid sequence SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:154, SEQ ID NO:160, SEQ ID NO:163, SEQ ID NO:169, SEQ ID NO:1862, SEQ ID NO:1868, SEQ ID NO:1873, SEQ ID NO:1879, SEQ ID NO:1883, SEQ ID NO:1888, SEQ ID NO:1897, SEQ ID NO:1902, SEQ ID NO:1915, SEQ ID NO:1920, SEQ ID NO:1925, and/or SEQ ID NO:1930; and proteins encoded by allelic variants of nucleic acid molecules encoding HMT and HNC proteins having amino acid sequences SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:154, SEQ ID NO:160, SEQ ID NO:163, SEQ ID NO:169, SEQ ID NO:1862, SEQ ID NO:1868, SEQ ID NO:1873, SEQ ID NO:1879, SEQ ID NO:1883, SEQ ID NO:1888, SEQ ID NO:1897, SEQ ID NO:1902, SEQ ID NO:1915, SEQ ID NO:1920, SEQ ID NO:1925, and/or SEQ ID NO:1930. Also preferred are fragments thereof having at least about 6 amino acid residues.
[0157] In one embodiment of the present invention,
[0158] In one embodiment, a preferred flea HMT or HNC protein comprises an amino acid sequence of at least about 35 amino acids, preferably at least about 50 amino acids, more preferably at least about 100 amino acids, more preferably at least about 200 amino acids, more preferably at least about 250 amino acids, more preferably at least about 300 amino acids, more preferably at least about 350 amino acids, more preferably at least about 400 amino acids, more preferably at least about 450 amino acids, more preferably at least about 500 amino acids, even more preferably at least about 550 amino acids, and even more preferably at least about 575 amino acids. In another embodiment, preferred flea HMT and HNC proteins comprise full-length proteins, i.e., proteins encoded by full-length coding regions, or post-translationally modified proteins thereof, such as mature proteins from which initiating methionine and/or signal sequences or “pro” sequences have been removed.
[0159] A fragment of an HMT and/or HNC protein of the present invention preferably comprises at least about 5 amino acids, more preferably at least about 10 amino acids, more preferably at least about 15 amino acids, more preferably at least about 20 amino acids, more preferably at least about 25 amino acids, more preferably at least about 30 amino acids, more preferably at least about 35 amino acids, more preferably at least about 40 amino acids, more preferably at least about 45 amino acids, more preferably at least about 50 amino acids, more preferably at least about 55 amino acids, more preferably at least about 60 amino acids, more preferably at least about 65 amino acids, more preferably at least about 70 amino acids, more preferably at least about 75 amino acids, more preferably at least about 80 amino acids, more preferably at least about 85 amino acids, more preferably at least about 90 amino acids, more preferably at least about 95 amino acids, and even more preferably at least about 100 amino acids in length.
[0160] Additional preferred HMT and HNC proteins of the present invention include proteins encoded by nucleic acid molecules comprising at least a portion of nCfALN
[0161] Also preferred are HMT and HNC proteins encoded by nucleic acid molecules having nucleic acid sequences comprising at least a portion of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:153, SEQ ID NO:156, SEQ ID NO:159, SEQ ID NO:162, SEQ ID NO:165, SEQ ID NO:168, SEQ ID NO:1859, SEQ ID NO:1861, SEQ ID NO:1864, SEQ ID NO:1867, SEQ ID NO:1870, SEQ ID NO:1872, SEQ ID NO:1875, SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1881, SEQ ID NO:1882, SEQ ID NO:1885, SEQ ID NO:1887, SEQ ID NO:1890, SEQ ID NO:1892, SEQ ID NO:1894, SEQ ID NO:1896, SEQ ID NO:1899, SEQ ID NO:1901, SEQ ID NO:1904, SEQ ID NO:1906, SEQ ID NO:1908, SEQ ID NO:1910, SEQ ID NO:1912, SEQ ID NO:1914, SEQ ID NO:1917, SEQ ID NO:1919, SEQ ID NO:1922, SEQ ID NO:1924, SEQ ID NO:1927, and/or SEQ ID NO:1929, as well as allelic variants of these nucleic acid molecules. A portion of such HMT and HNC nucleic acid molecule is preferably at least 18 nucleotides in length.
[0162] In another embodiment, a preferred flea HMT and/or HNC protein of the present invention is encoded by a nucleic acid molecule comprising at least about 15 nucleotides, more preferably at least about 18 nucleotides, more preferably at least about 20 nucleotides, more preferably at least about 25 nucleotides, more preferably at least about 30 nucleotides, more preferably at least about 40 nucleotides, more preferably at least about 50 nucleotides, more preferably at least about 100 nucleotides, more preferably at least about 150 nucleotides, more preferably at least about 350 nucleotides, more preferably at least about 450 nucleotides, more preferably at least about 550 nucleotides, more preferably at least about 650 nucleotides, more preferably at least about 750 nucleotides, more preferably at least about 1000 nucleotides, more preferably at least about 1500 nucleotides, more preferably at least about 1750 nucleotides more preferably at least about 2000 nucleotides, and even more preferably at least about 2250 nucleotides in length. Within this embodiment is a HMT protein encoded by at least a portion of nCfALN
[0163] Preferred flea HMT and HNC proteins of the present invention can be used to develop inhibitors that, when administered to an animal in an effective manner, are capable of protecting that animal from flea infestation. In accordance with the present invention, the ability of an inhibitor of the present invention to protect an animal from flea infestation refers to the ability of that protein to, for example, treat, ameliorate and/or prevent infestation caused by fleas. In particular, the phrase “to protect an animal from flea infestation” refers to reducing the potential for flea population expansion on and around the animal (i.e., reducing the flea burden). Preferably, the flea population size is decreased, optimally to an extent that the animal is no longer bothered by fleas. A host animal, as used herein, is an animal from which fleas can feed by attaching to and feeding through the skin of the animal. Fleas, and other ectoparasites, can live on a host animal for an extended period of time or can attach temporarily to an animal in order to feed. At any given time, a certain percentage of a flea population can be on a host animal whereas the remainder can be in the environment of the animal. Such an environment can include not only adult fleas, but also flea eggs and/or flea larvae. The environment can be of any size such that fleas in the environment are able to jump onto and off of a host animal. For example, the environment of an animal can include plants, such as crops, from which fleas infest an animal. As such, it is desirable not only to reduce the flea burden on an animal per se, but also to reduce the flea burden in the environment of the animal.
[0164] Suitable fleas to target include any flea that is essentially incapable of causing disease in an animal administered an inhibitor of the present invention. As such, fleas to target include any flea that produces a protein that can be targeted by an inhibitory compound that inhibits a flea HMT or HNC protein function, thereby resulting in the decreased ability of the parasite to cause disease in an animal. Preferred fleas to target include fleas of the following genera: Ctenocephalides, Cyopsyllus, Diamanus (Oropsylla), Echidnophaga, Nosopsyllus, Pulex, Tunga, and Xenopsylla, with those of the species
[0165] One embodiment of a flea HMT and/or HNC protein of the present invention is a fusion protein that includes a flea HMT and/or HNC protein-containing domain attached to one or more fusion segments. Suitable fusion segments for use with the present invention include, but are not limited to, segments that can: enhance a protein's stability; act as an immunopotentiator to enhance an immune response against a flea HMT and/or HNC protein; and/or assist in purification of a flea HMT and/or HNC protein (e.g., by affinity chromatography). A suitable fusion segment can be a domain of any size that has the desired function (e.g., imparts increased stability, imparts increased immunogenicity to a protein, and/or simplifies purification of a protein). Fusion segments can be joined to amino and/or carboxyl termini of the flea HMT-containing and/or HNC-containing domain of the protein and can be susceptible to cleavage in order to enable straight-forward recovery of a flea HMT and/or HNC protein. Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid molecule that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of an HMT-containing and/or HNC-containing domain. Preferred fusion segments include a metal binding domain (e.g., a poly-histidine segment); an immunoglobulin binding domain (e.g., Protein A; Protein G; T cell; B cell; Fc receptor or complement protein antibody-binding domains); a sugar binding domain (e.g., a maltose binding domain); and/or a “tag” domain (e.g., at least a portion of β-galactosidase, a strep tag peptide, a T7 tag peptide, a Flag™ peptide, or other domains that can be purified using compounds that bind to the domain, such as monoclonal antibodies). More preferred fusion segments include metal binding domains, such as a poly-histidine segment; a maltose binding domain; a strep tag peptide, such as that available from Biometra in Tampa, Fla.; and an S10 peptide.
[0166] The present invention also includes mimetopes of flea HMT and/or HNC proteins of the present invention. As used herein, a mimetope of a flea HMT and/or HNC protein of the present invention refers to any compound that is able to mimic the activity of such an HMT and/or HNC protein, often because the mimetope has a structure that mimics the particular HMT and/or HNC protein. Mimetopes can be, but are not limited to: peptides that have been modified to decrease their susceptibility to degradation such as all-D retro peptides; anti-idiotypic and/or catalytic antibodies, or fragments thereof; non-proteinaceous immunogenic portions of an isolated protein (e.g., carbohydrate structures); and synthetic or natural organic molecules, including nucleic acids. Such mimetopes can be designed using computer-generated structures of proteins of the present invention. Mimetopes can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides or other organic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner.
[0167] Another embodiment of the present invention is an isolated nucleic acid molecule comprising a flea HMT and/or HNC nucleic acid molecule, i.e. a nucleic acid molecule that can be isolated from a HMT cDNA library, from a HNC cDNA library, or from both libraries. As used herein, HMT and HNC nucleic acid molecules has the same meaning as HMT and/or HNC nucleic acid molecule. The identifying characteristics of such nucleic acid molecules are heretofore described. A nucleic acid molecule of the present invention can include an isolated natural flea HMT and/or HNC gene or a homologue thereof, the latter of which is described in more detail below. A nucleic acid molecule of the present invention can include one or more regulatory regions, full-length or partial coding regions, or combinations thereof. The minimal size of a nucleic acid molecule of the present invention is a size sufficient to allow the formation of a stable hybrid (i.e., hybridization under stringent hybridization conditions) with the complementary sequence of another nucleic acid molecule. As such, the minimal size of a HMT and/or HNC nucleic acid molecule of the present invention is from about 12 to about 18 nucleotides in length. Suitable and preferred fleas from which to isolate nucleic acid molecules of the present invention are disclosed herein. Particularly preferred HMT and/or HNC nucleic acid molecules include
[0168] In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subjected to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA. As such, “isolated” does not reflect the extent to which the nucleic acid molecule has been purified. Isolated flea HMT and/or HNC nucleic acid molecules of the present invention, or homologues thereof, can be isolated from a natural source or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification or cloning) or chemical synthesis. Isolated flea HMT and/or HNC nucleic acid molecules, and homologues thereof, can include, for example, natural allelic variants and nucleic acid molecules modified by nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode a HMT and/or HNC protein of the present invention.
[0169] A flea HMT and/or HNC nucleic acid molecule homologue can be produced using a number of methods known to those skilled in the art, see, for example, Sambrook et al., ibid., is incorporated by reference herein in its entirety. For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis and recombinant DNA techniques such as site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments, PCR amplification, synthesis of oligonucleotide mixtures and ligation of mixture groups to “build” a mixture of nucleic acid molecules, and combinations thereof. Nucleic acid molecule homologues can be selected by hybridization with flea HMT and/or HNC nucleic acid molecules or by screening the function of a protein encoded by the nucleic acid molecule (e.g., ability to elicit an immune response against at least one epitope of a flea HMT or HNC protein or to effect HMT or HNC activity).
[0170] An isolated nucleic acid molecule of the present invention can include a nucleic acid sequence that encodes at least one flea HMT or HNC protein of the present invention, examples of such proteins being disclosed herein. Although the phrase “nucleic acid molecule” primarily refers to the physical nucleic acid molecule and the phrase “nucleic acid sequence” primarily refers to the sequence of nucleotides on the nucleic acid molecule, the two phrases can be used interchangeably, especially with respect to a nucleic acid molecule, or a nucleic acid sequence, being capable of encoding a flea HMT or HNC protein.
[0171] A preferred nucleic acid molecule of the present invention, when administered to an animal, is capable of protecting that animal from flea infestation. As will be disclosed in more detail below, such a nucleic acid molecule can be, or encode, an antisense RNA, a molecule capable of triple helix formation, a ribozyme, or other nucleic acid-based drug compound. In additional embodiments, a nucleic acid molecule of the present invention can encode a protective protein (e.g., an HMT or HNC protein of the present invention), the nucleic acid molecule being delivered to the animal, for example, by direct injection (i.e, as a genetic vaccine) or in a vehicle such as a recombinant virus vaccine or a recombinant cell vaccine.
[0172] In one embodiment of the present invention, a preferred flea HMT and/or HNC nucleic acid molecule includes an isolated nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to about 30% base pair mismatch, more preferably under conditions that allow less than or equal to about 25% base pair mismatch, more preferably under conditions that allow less than or equal to about 20% base pair mismatch, more preferably under conditions that allow less than or equal to about 15% base pair mismatch, more preferably under conditions that allow less than or equal to about 10% base pair mismatch and even more preferably under conditions that allow less than or equal to about 5% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:1859, SEQ ID NO:1860, SEQ ID NO:1861, SEQ ID NO:1863, SEQ ID NO:1864, SEQ ID NO:1866, SEQ ID NO:1867, SEQ ID NO:1869, SEQ ID NO:1870, SEQ ID NO:1871, SEQ ID NO:1872, SEQ ID NO:1874, SEQ ID NO:1875, SEQ ID NO:1876, SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1880, SEQ ID NO:1881, SEQ ID NO:1882, SEQ ID NO:1884, SEQ ID NO:1885, SEQ ID NO:1886, SEQ ID NO:1887, SEQ ID NO:1889, SEQ ID NO:1890, SEQ ID NO:1891, SEQ ID NO:1892, SEQ ID NO:1893, SEQ ID NO:1894, SEQ ID NO:1895, SEQ ID NO:1896, SEQ ID NO:1898, SEQ ID NO:1899, SEQ ID NO:1900, SEQ ID NO:1901, SEQ ID NO:1903, SEQ ID NO:1904, SEQ ID NO:1905, SEQ ID NO:1906, SEQ ID NO:1907, SEQ ID NO:1908, SEQ ID NO:1909, SEQ ID NO:1910, SEQ ID NO:1911, SEQ ID NO:1912,SEQ ID NO:1913, SEQ ID NO:1914, SEQ ID NO:1916, SEQ ID NO:1917, SEQ ID NO:1918, SEQ ID NO:1919, SEQ ID NO:1921, SEQ ID NO:1922, SEQ ID NO:1923, SEQ ID NO:1924, SEQ ID NO:1926, SEQ ID NO:1927, SEQ ID NO:1928, SEQ ID NO:1929, and/or SEQ ID NO:1931, a nucleic acid molecule of Table I, Table II, Table III or Table IV and/or a nucleic acid molecule that is complementary to a nucleic acid molecule of Table I, Table II, Table m or Table IV.
[0173] Another embodiment of the present invention includes a HMT and/or HNC nucleic acid molecule, wherein said nucleic acid molecule hybridizes, in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:1859, SEQ ID NO:1860, SEQ ID NO:1861, SEQ ID NO:1863, SEQ ID NO:1864, SEQ ID NO:1866, SEQ ID NO:1867, SEQ ID NO:1869, SEQ ID NO:1870, SEQ ID NO:1871, SEQ ID NO:1872, SEQ ID NO:1874, SEQ ID NO:1875, SEQ ID NO:1876, SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1880, SEQ ID NO:1881, SEQ ID NO:1882, SEQ ID NO:1884, SEQ ID NO:1885, SEQ ID NO:1886, SEQ ID NO:1887, SEQ ID NO:1889, SEQ ID NO:1890, SEQ ID NO:1891, SEQ ID NO:1892, SEQ ID NO:1893, SEQ ID NO:1894, SEQ ID NO:1895, SEQ ID NO:1896, SEQ ID NO:1898, SEQ ID NO:1899, SEQ ID NO:1900, SEQ ID NO:1901, SEQ ID NO:1903, SEQ ID NO:1904, SEQ ID NO:1905, SEQ ID NO:1906, SEQ ID NO:1907, SEQ ID NO:1908, SEQ ID NO:1909, SEQ ID NO:1910, SEQ ID NO:1911, SEQ ID NO:1912, SEQ ID NO:1913, SEQ ID NO:1914, SEQ ID NO:1916, SEQ ID NO:1917, SEQ ID NO:1918, SEQ ID NO:1919, SEQ ID NO:1921, SEQ ID NO:1922, SEQ ID NO:1923, SEQ ID NO:1924, SEQ ID NO:1926, SEQ ID NO:1927, SEQ ID NO:1928, SEQ ID NO:1929, and/or SEQ ID NO:1931, a nucleic acid molecule of Table I, Table II, Table III or Table IV and/or a nucleic acid molecule that is complementary to a nucleic acid molecule of Table I, Table II, Table III or Table IV. Additional preferred nucleic acid molecules of the present invention include oligonucleotides of an isolated nucleic acid molecule, wherein said nucleic acid molecule hybridizes, in a solution comprising 1× SSC and 0% formamide, at a temperature of about 47.5° C., to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:1859, SEQ ID NO:1860, SEQ ID NO:1861, SEQ ID NO:1863, SEQ ID NO:1864, SEQ ID NO:1866, SEQ ID NO:1867, SEQ ID NO:1869, SEQ ID NO:1870, SEQ ID NO:1871, SEQ ID NO:1872, SEQ ID NO:1874, SEQ ID NO:1875, SEQ ID NO:1876, SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1880, SEQ ID NO:1881, SEQ ID NO:1882, SEQ ID NO:1884, SEQ ID NO:1885, SEQ ID NO:1886, SEQ ID NO:1887, SEQ ID NO:1889, SEQ ID NO:1890, SEQ ID NO:1891, SEQ ID NO:1892, SEQ ID NO:1893, SEQ ID NO:1894 SEQ ID NO:1895, SEQ ID NO:1896, SEQ ID NO:1898, SEQ ID NO:1899, SEQ ID NO:1900, SEQ ID NO:1901, SEQ ID NO:1903, SEQ ID NO:1904, SEQ ID NO:1905, SEQ ID NO:1906, SEQ ID NO:1907, SEQ ID NO:1908, SEQ ID NO:1909, SEQ ID NO:1910, SEQ ID NO:1911, SEQ ID NO:1912, SEQ ID NO:1913, SEQ ID NO:1914, SEQ ID NO:1916, SEQ ID NO:1917, SEQ ID NO:1918, SEQ ID NO:1919, SEQ ID NO:1921, SEQ ID NO:1922, SEQ ID NO:1923, SEQ ID NO:1924, SEQ ID NO:1926, SEQ ID NO:1927, SEQ ID NO:1928, SEQ ID NO:1929, and/or SEQ ID NO:1931, a nucleic acid molecule of Table I, Table II, Table III or Table IV and/or a nucleic acid molecule that is complementary to a nucleic acid molecule of Table I, Table II, Table m or Table IV, wherein said oligonucleotide comprises at least about 18 nucleotides.
[0174] Additional preferred flea HMT and/or HNC nucleic acid molecules of the present invention include nucleic acid molecules comprising a nucleic acid sequence that is preferably at least about 70%, more preferably at least about 75%, more preferably at least about 80% more preferably at least about 85%, more preferably at least about 90%, and even more preferably at least about 95% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO :18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:1859, SEQ ID NO:1860, SEQ ID NO:1861, SEQ ID NO:1863, SEQ ID NO:1864, SEQ ID NO:1866, SEQ ID NO:1867, SEQ ID NO:1869, SEQ ID NO:1870, SEQ ID NO:1871, SEQ ID NO:1872, SEQ ID NO:1874, SEQ ID NO:1875, SEQ ID NO:1876, SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1880, SEQ ID NO:1881, SEQ ID NO:1882, SEQ ID NO:1884, SEQ ID NO:1885, SEQ ID NO:1886, SEQ ID NO:1887, SEQ ID NO:1889, SEQ ID NO:1890, SEQ ID NO:1891, SEQ ID NO:1892, SEQ ID NO:1893, SEQ ID NO:1894, SEQ ID NO:1895, SEQ ID NO:1896, SEQ ID NO:1898, SEQ ID NO:1899, SEQ ID NO:1900, SEQ ID NO:1901, SEQ ID NO:1903, SEQ ID NO:1904, SEQ ID NO:1905, SEQ ID NO:1906, SEQ ID NO:1907, SEQ ID NO:1908, SEQ ID NO:1909, SEQ ID NO:1910,SEQ ID NO:1911, SEQ ID NO:1912,SEQ ID NO:1913, SEQ ID NO:1914, SEQ ID NO:1916, SEQ ID NO:1917, SEQ ID NO:1918, SEQ ID NO:1919, SEQ ID NO:1921, SEQ ID NO:1922, SEQ ID NO:1923, SEQ ID NO:1924, SEQ ID NO:1926, SEQ ID NO:1927, SEQ ID NO:1928, SEQ ID NO:1929, and/or SEQ ID NO:1931, a nucleic acid molecule of Table I, Table II, Table III or Table IV and/or a nucleic acid molecule that is complementary to a nucleic acid molecule of Table I, Table II, Table III or Table IV. Also preferred are oligonucleotides of any of such nucleic acid molecules. Percent identity may be determined using the GCG™ Wisconsin Package Version 9.0 sequence analysis software, using default parameters.
[0175] One embodiment of the present invention is a nucleic acid molecule comprising all or part of nucleic acid molecules nCfALN
[0176] In one embodiment, HMT and/or HNC nucleic acid molecule of the present invention encodes a protein that is at least about 70%, preferably at least about 75%, more preferably at least about 80%, even more preferably at least about 85%, even more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 98%, and even more preferably at least about 100% identical to PCfALN
[0177] In one embodiment, a HMT and/or HNC nucleic acid molecule of the present invention encodes a protein having an amino acid sequence that is at least about 70%, preferably at least about 75%, more preferably at least about 80%, even more preferably at least about 85%, even more preferably at least about 90%, even more preferably at least about 95%, even more preferably at least about 98%, and even more preferably at least about 100% identical to SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:154, SEQ ID NO:160, SEQ ID NO:163, SEQ ID NO:169, SEQ ID NO:1862, SEQ ID NO:1868, SEQ ID NO:1873, SEQ ID NO:1879, SEQ ID NO:1883, SEQ ID NO:1888, SEQ ID NO:1897, SEQ ID NO:1902, SEQ ID NO:1915, SEQ ID NO:1920, SEQ ID NO:1925, and/or SEQ ID NO:1930 and/or a protein encoded by a nucleic acid molecule having a sequence of Table I, Table II, Table III and/or Table IV. The present invention also includes a HMT and/or HNC nucleic acid molecule encoding a protein having at least a portion of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:154, SEQ ID NO:160, SEQ ID NO:163, SEQ ID NO:169, SEQ ID NO:1862, SEQ ID NO:1868, SEQ ID NO:1873, SEQ ID NO:1879, SEQ ID NO:1883, SEQ ID NO:1888, SEQ ID NO:1897, SEQ ID NO:1902, SEQ ID NO:1915, SEQ ID NO:1920, SEQ ID NO:1925, and/or SEQ ID NO:1930 and/or a protein encoded by a nucleic acid molecule having a sequence of Table I, Table II, Table m and/or Table IV, as well as allelic variants of a nucleic acid molecule encoding a protein having these sequences, including nucleic acid molecules that have been modified to accommodate codon usage properties of the cells in which such nucleic acid molecules are to be expressed.
[0178] In another embodiment, a preferred flea HMT and/or HNC nucleic acid molecule of the present invention comprises a nucleic acid molecule comprising at least about 15 nucleotides, more preferably at least about 18 nucleotides, more preferably at least about 20 nucleotides, more preferably at least about 25 nucleotides, more preferably at least about 30 nucleotides, more preferably at least about 40 nucleotides, more preferably at least about 50 nucleotides, more preferably at least about 100 nucleotides, more preferably at least about 150 nucleotides, more preferably at least about 350 nucleotides, more preferably at least about 450 nucleotides, more preferably at least about 550 nucleotides, more preferably at least about 650 nucleotides, more preferably at least about 750 nucleotides, more preferably at least about 1000 nucleotides, more preferably at least about 1500 nucleotides, more preferably at least about 1750 nucleotides more preferably at least about 2000 nucleotides, and even more preferably at least about 2250 nucleotides in length.
[0179] In another embodiment, a preferred flea HMT and/or HNC nucleic acid molecule encodes a protein comprising at least about 5 amino acids, preferably at least about 6 amino acids, more preferably at least about 10 amino acids, more preferably at least about 15 amino acids, more preferably at least about 20 amino acids, more preferably at least about 25 amino acids, more preferably at least about 30 amino acids, more preferably at least about 40 amino acids, more preferably at least about 50 amino acids, more preferably at least about 100 amino acids, more preferably at least about 150 amino acids, more preferably at least about 200 amino acids, more preferably at least about 300 amino acids, more preferably at least about 400 amino acids, more preferably at least about 500 amino acids, even more preferably at least about 560 amino acids in length.
[0180] In another embodiment, a preferred flea HMT and/or HNC nucleic acid molecule of the present invention comprises an apparently full-length HMT and/or HNC coding region, i.e., the preferred nucleic acid molecule encodes an apparently full-length HMT and/or HNC protein, or a post-translationally modified protein thereof. In one embodiment, a preferred HMT and/or HNC nucleic acid molecule of the present invention encodes a mature protein.
[0181] In another embodiment, a preferred flea HMT and/or HNC nucleic acid molecule of the present invention comprises a nucleic acid molecule comprising SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:1859, SEQ ID NO:1860, SEQ ID NO:1861, SEQ ID NO:1863, SEQ ID NO:1864, SEQ ID NO:1866, SEQ ID NO:1867, SEQ ID NO:1869, SEQ ID NO:1870, SEQ ID NO:1871, SEQ ID NO:1872, SEQ ID NO:1874, SEQ ID NO:1875, SEQ ID NO:1876, SEQ ID NO:1877, SEQ ID NO:1878, SEQ ID NO:1880,SEQ ID NO:1881, SEQ ID NO:1882, SEQ ID NO:1884, SEQ ID NO:1885, SEQ ID NO:1886, SEQ ID NO:1887, SEQ ID NO:1889, SEQ ID NO:1890, SEQ ID NO:1891, SEQ ID NO:1892, SEQ ID NO:1893, SEQ ID NO:1894, SEQ ID NO:1895, SEQ ID NO:1896, SEQ ID NO:1898, SEQ ID NO:1899, SEQ ID NO:1900, SEQ ID NO:1901, SEQ ID NO:1903, SEQ ID NO:1904, SEQ ID NO:1905, SEQ ID NO:1906, SEQ ID NO:1907, SEQ ID NO:1908, SEQ ID NO:1909, SEQ ID NO:1910, SEQ ID NO:1911, SEQ ID NO:1912, SEQ ID NO:1913, SEQ ID NO:1914, SEQ ID NO:1916, SEQ ID NO:1917, SEQ ID NO:1918, SEQ ID NO:1919, SEQ ID NO:1921, SEQ ID NO:1922, SEQ ID NO:1923, SEQ ID NO:1924, SEQ ID NO:1926, SEQ ID NO:1927,SEQ ID NO:1928,SEQ ID NO:1929, and/or SEQ ID NO:1931.
[0182] Knowing the nucleic acid sequences of certain flea HMT and/or HNC nucleic acid molecules of the present invention allows one skilled in the art to, for example, (a) make copies of those nucleic acid molecules, (b) obtain nucleic acid molecules including at least a portion of such nucleic acid molecules (e.g., nucleic acid molecules including full-length genes, full-length coding regions, regulatory control sequences, truncated coding regions), and (c) obtain other flea HMT and/or HNC nucleic acid molecules. Such nucleic acid molecules can be obtained in a variety of ways including screening appropriate expression libraries with antibodies of the present invention; traditional cloning techniques using oligonucleotide probes of the present invention to screen appropriate libraries; and PCR amplification of appropriate libraries or DNA using oligonucleotide primers of the present invention. Preferred libraries to screen or from which to amplify nucleic acid molecules include flea 1
[0183] The present invention also includes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent hybridization conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the present invention such as those comprising
[0184] One embodiment of the present invention includes a recombinant vector, which includes at least one isolated nucleic acid molecule of the present invention, inserted into any vector capable of delivering the nucleic acid molecule into a host cell. Such a vector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are derived from a species other than the species from which the nucleic acid molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of flea HMT and/or HNC nucleic acid molecules of the present invention.
[0185] One type of recombinant vector, referred to herein as a recombinant molecule, comprises a nucleic acid molecule of the present invention operatively linked to an expression vector. The phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified nucleic acid molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, parasite, insect, other animal, and plant cells. Preferred expression vectors of the present invention can direct gene expression in bacterial, yeast, insect and mammalian cells, and more preferably in the cell types disclosed herein.
[0186] In particular, expression vectors of the present invention contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention. In particular, recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are sequences that control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those that function in bacterial, yeast, or insect and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (such as lambda p
[0187] Suitable and preferred nucleic acid molecules to include in recombinant vectors of the present invention are as disclosed herein. Preferred nucleic acid molecules to include in recombinant vectors, and particularly in recombinant molecules, include nucleic acid molecules having a sequence of Table I, Table II, Table III and/or Table IV. Particularly preferred nucleic acid molecules to include in recombinant vectors, and particularly in recombinant molecules, include nCfALN
[0188] Recombinant molecules of the present invention may also (a) contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed flea protein of the present invention to be secreted from the cell that produces the protein and/or (b) contain fusion sequences which lead to the expression of nucleic acid molecules of the present invention as fusion proteins. Examples of suitable signal segments include any signal segment capable of directing the secretion of a protein of the present invention. Preferred signal segments include, but are not limited to, tissue plasminogen activator (t-PA), interferon, interleukin, growth hormone, histocompatibility and viral envelope glycoprotein signal segments. Suitable fusion segments encoded by fusion segment nucleic acids are disclosed herein. In addition, a nucleic acid molecule of the present invention can be joined to a fusion segment that directs the encoded protein to the proteosome, such as a ubiquitin fusion segment. Eukaryotic recombinant molecules may also include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules of the present invention.
[0189] Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. It is to be noted that a cell line refers to any recombinant cell of the present invention that is not a transgenic animal. Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained. Preferred nucleic acid molecules with which to transform a cell include
[0190] Suitable host cells to transform include any cell that can be transformed with a nucleic acid molecule of the present invention. Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule (e.g., nucleic acid molecules encoding one or more proteins of the present invention and/or other proteins useful in the production of multivalent vaccines). Host cells of the present invention either can be endogenously (i.e., naturally) capable of producing flea HMT and/or HNC proteins of the present invention or can be capable of producing such proteins after being transformed with at least one nucleic acid molecule of the present invention. Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include bacterial, fungal (including yeast), parasite (including helminth, protozoa and ectoparasite), other insect, other animal and plant cells. Preferred host cells include bacterial, mycobacterial, yeast, insect and mammalian cells. More preferred host cells include Salmonella, Escherichia, Bacillus, Caulobacter, Listeria, Saccharomyces, Pichia, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells (Madin-Darby canine kidney cell line), CRFK cells (Crandell feline kidney cell line), CV-1 cells (African monkey kidney cell line used, for example, to culture raccoon poxvirus), COS (e.g., COS-7) cells, and Vero cells. Particularly preferred host cells are
[0191] A recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences, examples of which are disclosed herein. The phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell.
[0192] A recombinant cell of the present invention includes any cell transformed with at least one of any nucleic acid molecule of the present invention. Suitable and preferred nucleic acid molecules as well as suitable and preferred recombinant molecules with which to transfer cells are disclosed herein.
[0193] Recombinant cells of the present invention can also be co-transformed with one or more recombinant molecules including flea HMT and/or HNC nucleic acid molecules encoding one or more proteins of the present invention and one or more other nucleic acid molecules encoding other protective compounds, as disclosed herein (e.g., to produce multivalent vaccines).
[0194] Recombinant DNA technologies can be used to improve expression of transformed nucleic acid molecules by manipulating, for example, the number of copies of the nucleic acid molecules within a host cell, the efficiency with which those nucleic acid molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of nucleic acid molecules of the present invention include, but are not limited to, operatively linking nucleic acid molecules to high-copy number plasmids, integration of the nucleic acid molecules into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of nucleic acid molecules of the present invention to correspond to the codon usage of the host cell, deletion of sequences that destabilize transcripts, and use of control signals that temporally separate recombinant cell growth from recombinant enzyme production during fermentation. The activity of an expressed recombinant protein of the present invention may be improved by fragmenting, modifying, or derivatizing nucleic acid molecules encoding such a protein.
[0195] Isolated flea HMT and/or HNC proteins of the present invention can be produced in a variety of ways, including production and recovery of natural proteins, production and recovery of recombinant proteins, and chemical synthesis of the proteins. In one embodiment, an isolated protein of the present invention is produced by culturing a cell capable of expressing the protein under conditions effective to produce the protein, and recovering the protein. A preferred cell to culture is a recombinant cell of the present invention. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An effective, medium refers to any medium in which a cell is cultured to produce a flea HMT and/or HNC protein of the present invention. Such medium typically comprises an aqueous medium having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. Cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes, and petri plates. Culturing can be carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. Such culturing conditions are within the expertise of one of ordinary skill in the art. Examples of suitable conditions are included in the Examples section.
[0196] Depending on the vector and host system used for production, resultant proteins of the present invention may either remain within the recombinant cell; be secreted into the fermentation medium; be secreted into a space between two cellular membranes, such as the periplasmic space in
[0197] The phrase “recovering the protein”, as well as similar phrases, refers to collecting the whole fermentation medium containing the protein and need not imply additional steps of separation or purification. Proteins of the present invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization. Proteins of the present invention are preferably retrieved in “substantially pure” form. As used herein, “substantially pure” refers to a purity that allows for the effective use of the protein as a therapeutic composition or diagnostic. A therapeutic composition for animals, for example, should exhibit no substantial toxicity and preferably should be capable of stimulating the production of antibodies in a treated animal.
[0198] The present invention also includes isolated (i.e., removed from their natural milieu) antibodies that selectively bind to a flea HMT and/or HNC protein of the present invention or a mimetope thereof (e.g., anti-
[0199] Isolated antibodies of the present invention can include antibodies in serum, or antibodies that have been purified to varying degrees. Antibodies of the present invention can be polyclonal or monoclonal, or can be functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies or chimeric antibodies that can bind to one or more epitopes.
[0200] A preferred method to produce antibodies of the present invention includes (a) administering to an animal an effective amount of a protein, peptide or mimetope thereof of the present invention to produce the antibodies and (b) recovering the antibodies. In another method, antibodies of the present invention are produced recombinantly using techniques as heretofore disclosed to produce HMT and/or HNC proteins of the present invention. Antibodies raised against defined proteins or mimetopes can be advantageous because such antibodies are not substantially contaminated with antibodies against other substances that might otherwise cause interference in a diagnostic assay or side effects if used in a therapeutic composition.
[0201] Antibodies of the present invention have a variety of potential uses that are within the scope of the present invention. For example, such antibodies can be used (a) as therapeutic compounds to passively immunize an animal in order to protect the animal from fleas susceptible to treatment by such antibodies and/or (b) as tools to screen expression libraries and/or to recover desired proteins of the present invention from a mixture of proteins and other contaminants. Furthermore, antibodies of the present invention can be used to target cytotoxic agents to fleas in order to directly kill such fleas. Targeting can be accomplished by conjugating (i.e., stably joining) such antibodies to the cytotoxic agents using techniques known to those skilled in the art. Suitable cytotoxic agents are known to those skilled in the art.
[0202] One embodiment of the present invention is a therapeutic composition that, when administered to an animal susceptible to flea infestation, is capable of protecting that animal from flea infestation. Therapeutic compositions of the present invention include at least one of the following protective molecules: an isolated flea HMT and/or HNC protein; a mimetope of an isolated flea HMT and/or HNC protein; an isolated flea HMT and/or HNC nucleic acid molecule; and/or a compound derived from said isolated flea HMT and/or HNC protein that inhibits HMT and/or HNC protein activity. A therapeutic composition of the present invention can further comprise a component selected from the group of an excipient, a carrier, and/or an adjuvant; these components are described further herein. As used herein, a protective molecule or protective compound refers to a compound that, when administered to an animal in an effective manner, is able to treat, ameliorate, and/or prevent flea infestation. Preferred fleas to target are heretofore disclosed. One example of a protective molecule is a vaccine, such as, but not limited to, a naked nucleic acid vaccine, a recombinant virus vaccine, a recombinant cell vaccine, and a recombinant protein vaccine. Another example of a protective molecule is a compound that inhibits HMT and/or HNC protein activity, such as an isolated antibody that selectively binds to a flea HMT and/or HNC protein, a substrate analog of a flea HMT and/or HNC protein, anti-sense-, triplex formation-, ribozyme-, and/or RNA drug-based compounds, or other inorganic or organic molecules that inhibit HMT and/or HNC protein activity. Inhibiting flea HMT and/or HNC protein activity can refer to the ability of a compound to reduce the activity of flea HMT and/or HNC proteins. Inhibiting flea HMT and/or HNC protein activity can also refer to the ability of a compound to reduce the amount of flea HMT and/or HNC protein in a flea.
[0203] Another embodiment of the present invention includes a method to reduce a flea infestation in an animal susceptible to flea infestation. Such a method includes the step of administering to the animal a therapeutic molecule comprising a protective compound selected from the group consisting of (a) an isolated flea HMT and/or HNC protein; (b) a mimetope of an isolated flea HMT and/or HNC protein; (c) an isolated flea HMT and/or HNC nucleic acid molecule; and (d) a compound derived from an isolated flea HMT and/or HNC protein that inhibits HMT and/or HNC protein activity.
[0204] Therapeutic compositions of the present invention can be administered to any animal susceptible to flea infestation, preferably to mammals, and more preferably to dogs, cats, humans, ferrets, horses, cattle, sheep, and other pets, economic food animals, work animals and/or zoo animals. Preferred animals to protect against flea infestation include dogs, cats, humans, and ferrets, with dogs and cats being particularly preferred.
[0205] As used herein, the term derived, or the term derived from, refers to a peptide, antibody, mimetope, nucleic acid molecule, or other compound that was obtained from a flea HMT and/or HNC protein or nucleic acid molecule of the present invention. Methods to obtain derivatives from a HMT and/or HNC molecule of the present invention are known in the art, and as such include, but are not limited to molecular modeling of HMT and/or HNC proteins to determine active sites, i.e. sites that interact with other molecules, and predicting from these active sites smaller fragments and/or mimetopes that retain and/or mimic these active sites, thereby inhibiting HMT and/or HNC protein activity; screening of peptide or small chemical compound libraries against HMT and/or HNC proteins of the present invention; and screening of polyclonal or monoclonal antibodies to find antibodies that specifically bind HMT and/or HNC proteins of the present invention.
[0206] A HMT and/or HNC protein inhibitor of the present invention is identified by its ability to bind to, modify, or otherwise interact with, a flea HMT and/or HNC protein, thereby inhibiting the activity of HMT and/or HNC proteins. Suitable inhibitors of HMT and/or HNC protein activity are compounds that inhibit HMT and/or HNC protein activity in at least one of a variety of ways: (a) by binding to or otherwise interacting with or otherwise modifying HMT and/or HNC protein sites; (b) by binding to or otherwise interacting with or otherwise modifying the HMT and/or HNC protein active site; (c) by binding to the HMT and/or HNC protein and thus reducing the availability of the HMT and/or HNC protein in solution; and (d) by interacting with other regions of the HMT and/or HNC protein to inhibit HMT and/or HNC protein activity, for example, by allosteric interaction.
[0207] Flea HMT and/or HNC protein inhibitors can be used directly as compounds in compositions of the present invention to treat animals as long as such compounds are not harmful to host animals being treated. Preferred HMT and/or HNC protein inhibitors of the present invention include, but are not limited to, flea HMT and/or HNC protein substrate analogs, and other molecules that bind to a flea HMT and/or HNC proteins (e.g., to an allosteric site) in such a manner that the activity of the flea HMT and/or HNC protein is inhibited. A HMT and/or HNC protein substrate analog refers to a compound that interacts with (e.g., binds to, associates with, modifies) the active site of a HMT and/or HNC protein. A preferred HMT and/or HNC protein substrate analog inhibits HMT and/or HNC protein activity. HMT and/or HNC protein substrate analogs can be of any inorganic or organic composition. HMT and/or HNC protein substrate analogs can be, but need not be, structurally similar to a HMT and/or HNC protein natural substrate as long as they can interact with the active site of that HMT and/or HNC protein. HMT and/or HNC protein substrate analogs can be designed using computer-generated structures of HMT and/or HNC proteins of the present invention or computer structures of HMT and/or HNC protein's natural substrates. Preferred sites to model include one or more of the active sites of HMT and/or HNC protein. Substrate analogs can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides, peptidomimetic compounds, or other inorganic or organic molecules, and screening such samples for their ability to interfere with interaction between HMT and/or HNC proteins and their substrates, e.g. by affinity chromatography techniques. A preferred HMT and/or HNC protein substrate analog is a HMT and/or HNC protein mimetic compound, i.e., a compound that is structurally and/or functionally similar to a natural substrate of a HMT and/or HNC protein of the present invention, particularly to the region of the substrate that interacts with the HMT and/or HNC protein active site, but that inhibits HMT and/or HNC protein activity upon interacting with the HMT and/or HNC protein active site.
[0208] The present invention also includes a therapeutic composition comprising at least one protective molecule of the present invention in combination with at least one additional compound protective against one or more infectious agents.
[0209] In one embodiment, a therapeutic composition of the present invention can be used to protect an animal from flea infestation by administering such composition to a flea in order to prevent infestation. Such administration to the flea and/or animal could be oral, or by application to the animal's body surface (e.g. topical spot-on, or spraying onto the animal), or by application to the environment (e.g., spraying). Examples of such compositions include, but are not limited to, transgenic vectors capable of producing at least one therapeutic composition of the present invention. In another embodiment a flea can ingest therapeutic compositions, or products thereof, present on the surface of or in the blood of a host animal that has been administered a therapeutic composition of the present invention.
[0210] In accordance with the present invention, a host animal (i.e., an animal that is or is capable of being infested with fleas) is treated by administering to the animal a therapeutic composition of the present invention in such a manner that the composition itself (e.g., a HMT and/or HNC protein inhibitor, a HMT and/or HNC protein synthesis suppressor (i.e., a compound that decreases the production or half-life of a HMT and/or HNC protein in fleas), a HMT and/or HNC protein mimetope, or a anti-HMT and/or HNC antibody) or a product generated by the animal in response to administration of the composition (e.g., antibodies produced in response to administration of a flea HMT and/or HNC protein or nucleic acid molecule, or conversion of an inactive inhibitor “prodrug” to an active HMT and/or HNC protein inhibitor) ultimately enters the flea. A host animal is preferably treated in such a way that the compound or product thereof is present on the body surface of the animal or enters the blood stream of the animal. Fleas are then exposed to the composition or product when they feed from the animal. For example, flea HMT and/or HNC protein inhibitors administered to an animal are administered in such a way that the inhibitors enter the blood stream of the animal, where they can be taken up by feeding fleas.
[0211] The present invention also includes the ability to reduce larval flea infestation in that when fleas feed from a host animal that has been administered a therapeutic composition of the present invention, at least a portion of compounds of the present invention, or products thereof, in the blood taken up by the fleas are excreted by the fleas in feces, which is subsequently ingested by flea larvae. In particular, it is of note that flea larvae obtain most, if not all, of their nutrition from flea feces.
[0212] In accordance with the present invention, reducing HMT and/or HNC protein activity in a flea can lead to a number of outcomes that reduce flea burden on treated animals and their surrounding environments. Such outcomes include, but are not limited to, (a) reducing the viability of fleas that feed from the treated animal, (b) reducing the fecundity of female fleas that feed from the treated animal, (c) reducing the reproductive capacity of male fleas that feed from the treated animal, (d) reducing the viability of eggs laid by female fleas that feed from the treated animal, (e) altering the blood feeding behavior of fleas that feed from the treated animal (e.g., fleas take up less volume per feeding or feed less frequently), (f) reducing the viability of flea larvae, for example due to the feeding of larvae from feces of fleas that feed from the treated animal, (g) altering the development of flea larvae (e.g., by decreasing feeding behavior, inhibiting growth, inhibiting (e.g., slowing or blocking) molting, and/or otherwise inhibiting maturation to adults), and/or (h) altering or decreasing the ability of fleas or flea larvae to digest a blood meal.
[0213] In order to protect an animal from flea infestation, a therapeutic composition of the present invention is administered to the animal in an effective manner such that the composition is capable of protecting that animal from flea infestation. Therapeutic compositions of the present invention can be administered to animals prior to infestation in order to prevent infestation (i.e., as a preventative vaccine) and/or can be administered to animals after infestation. For example, proteins, mimetopes thereof, and antibodies thereof can be used as immunotherapeutic agents.
[0214] Therapeutic compositions of the present invention can be formulated in an excipient that the animal to be treated can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, or o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.
[0215] In one embodiment of the present invention, a therapeutic composition can include an adjuvant. Adjuvants are agents that are capable of enhancing the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, cytokines, chemokines, and compounds that induce the production of cytokines and chemokines (e.g., granulocyte macrophage colony stimulating factor (GM-CSF), Flt-3 ligand, granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), colony stimulating factor (CSF), erythropoietin (EPO), interleukin 2 (IL-2), interleukin-3 (IL-3), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 6 (IL-6), interleukin 7 (IL-7), interleukin 8 (IL-8), interleukin 10 (IL-10), interleukin 12 (IL-12), interferon gamma, interferon gamma inducing factor I (IGIF), transforming growth factor beta, RANTES (regulated upon activation, normal T cell expressed and presumably secreted), macrophage inflammatory proteins (e.g., MIP-1 alpha and MIP-1 beta), and Leishmania elongation initiating factor (LEIF)); bacterial components (e.g., endotoxins, in particular superantigens, exotoxins and cell wall components); aluminum-based salts; calcium-based salts; silica; polynucleotides; toxoids; serum proteins, viral coat proteins; block copolymer adjuvants (e.g., Hunter's Titermax™ adjuvant (Vaxcel™, Inc. Norcross, Ga.), Ribi adjuvants (Ribi ImmunoChem Research, Inc., Hamilton, Mont.); and saponins and their derivatives (e.g., Quil A (Superfos Biosector A/S, Denmark). Protein adjuvants of the present invention can be delivered in the form of the protein themselves or of nucleic acid molecules encoding such proteins using the methods described herein.
[0216] In one embodiment of the present invention, a therapeutic composition can include a carrier. Carriers include compounds that increase the half-life of a therapeutic composition in the treated animal. Suitable carriers include, but are not limited to, polymeric controlled release vehicles, biodegradable implants, liposomes, bacteria, viruses, other cells, oils, esters, and glycols.
[0217] One embodiment of the present invention is a controlled release formulation that is capable of slowly releasing a composition of the present invention into an animal. As used herein, a controlled release formulation comprises a composition of the present invention in a controlled release vehicle. Suitable controlled release vehicles include, but are not limited to, biocompatible polymers, other polymeric matrices, capsules, microcapsules, microparticles, bolus preparations, osmotic pumps, diffusion devices, liposomes, lipospheres, and transdermal delivery systems. Other controlled release formulations of the present invention include liquids that, upon administration to an animal, form a solid or a gel in situ. Preferred controlled release formulations are biodegradable (i.e., bioerodible).
[0218] A preferred controlled release formulation of the present invention is capable of releasing a composition of the present invention into the blood of the treated animal at a constant rate sufficient to attain therapeutic dose levels of the composition to protect an animal from flea infestation. The therapeutic composition is preferably released over a period of time ranging from about 1 to about 12 months. A controlled release formulation of the present invention is capable of effecting a treatment preferably for at least about 1 month, more preferably for at least about 3 months, even more preferably for at least about 6 months, even more preferably for at least about 9 months, and even more preferably for at least about 12 months.
[0219] Acceptable protocols to administer therapeutic compositions in an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art. A suitable single dose is a dose that is capable of protecting an animal from disease when administered one or more times over a suitable time period. For example, a preferred single dose of a protein, mimetope or antibody therapeutic composition, including a recombinant protein vaccine, is from about 1 microgram (jig) to about 10 milligrams (mg) of the therapeutic composition per kilogram body weight of the animal. Booster vaccinations can be administered from about 2 weeks to several years after the original administration. Booster administrations preferably are administered when the immune response of the animal becomes insufficient to protect the animal from disease. A preferred administration schedule is one in which from about 10 μg to about 1 mg of the therapeutic composition per kg body weight of the animal is administered from about one to about two times over a time period of from about 2 weeks to about 12 months. Modes of administration can include, but are not limited to, subcutaneous, intradermal, intravenous, intranasal, oral, transdermal, intraocular, intranasal, conjunctival, and intramuscular routes. Methods of administration for other therapeutic compounds can be determined by one skilled in the art, and may include administration of a therapeutic composition one or more times, on a daily, weekly, monthly or yearly regimen; routes of administration can be determined by one skilled in the art, and may include any route. A preferred route of administration of an inhibitory compound when administering to fleas is a topical, or “spot-on” formulation administered to the body surface of the animal, so that a flea would encounter the inhibitory compound when attached to the animal; another preferred route of administration of an inhibitory compound is an oral formulation that, when fed to an animal, would enter the bloodstream of the animal, which would then be transferred to a flea while feeding from the animal.
[0220] A recombinant protein vaccine of the present invention comprises a recombinantly-produced flea HMT and/or HNC protein of the present invention that is administered to an animal according to a protocol that results in the animal producing a sufficient immune response to protect itself from a flea infestation. Such protocols can be determined by those skilled in the art.
[0221] According to one embodiment, a nucleic acid molecule of the present invention can be administered to an animal in a fashion to enable expression of that nucleic acid molecule into a protective protein or protective RNA (e.g., antisense RNA, ribozyme, triple helix forms or RNA drug) in the animal. Nucleic acid molecules can be delivered to an animal in a variety of methods including, but not limited to, (a) administering a naked (i.e., not packaged in a viral coat or cellular membrane) nucleic acid as a genetic vaccine (e.g., as naked DNA or RNA molecules, such as is taught, for example in Wolff et al., 1990, Science 247, 1465-1468) or (b) administering a nucleic acid molecule packaged as a recombinant virus vaccine or as a recombinant cell vaccine (i.e., the nucleic acid molecule is delivered by a viral or cellular vehicle).
[0222] A genetic (i.e., naked nucleic acid) vaccine of the present invention includes a nucleic acid molecule of the present invention and preferably includes a recombinant molecule of the present invention that preferably is replication, or otherwise amplification, competent. A genetic vaccine of the present invention can comprise one or more nucleic acid molecules of the present invention in the form of, for example, a dicistronic recombinant molecule. Preferred genetic vaccines include at least a portion of a viral genome, i.e., a viral vector. Preferred viral vectors include those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, picornaviruses, and retroviruses, with those based on alphaviruses, such as sindbis or Semliki forest virus, species-specific herpesviruses and poxviruses being particularly preferred. Any suitable transcription control sequence can be used, including those disclosed as suitable for protein production. Particularly preferred transcription control sequences include cytomegalovirus immediate early (preferably in conjunction with Intron-A), Rous sarcoma virus long terminal repeat, and tissue-specific transcription control sequences, as well as transcription control sequences endogenous to viral vectors if viral vectors are used. The incorporation of a “strong” polyadenylation signal is also preferred.
[0223] Genetic vaccines of the present invention can be administered in a variety of ways, with intramuscular, subcutaneous, intradermal, transdermal, conjunctival, intraocular, intranasal and oral routes of administration being preferred. A preferred single dose of a genetic vaccine ranges from about 1 nanogram (ng) to about 600 μg, depending on the route of administration and/or method of delivery, as can be determined by those skilled in the art. Suitable delivery methods include, for example, by injection, as drops, aerosolized and/or topically. Genetic vaccines of the present invention can be contained in an aqueous excipient (e.g., phosphate buffered saline) alone or in a carrier (e.g., lipid-based vehicles).
[0224] A recombinant virus vaccine of the present invention includes a recombinant molecule of the present invention that is packaged in a viral coat and that can be expressed in an animal after administration. Preferably, the recombinant molecule is packaging- or replication-deficient and/or encodes an attenuated virus. A number of recombinant viruses can be used, including, but not limited to, those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, picornaviruses, and retroviruses. Preferred recombinant virus vaccines are those based on alphaviruses (such as Sindbis virus), raccoon poxviruses, species-specific herpesviruses and species-specific poxviruses. An example of methods to produce and use alphavirus recombinant virus vaccines are disclosed in U.S. Pat. No. 5,766,602 to Xiong and Grieve, which is incorporated by reference herein in its entirety.
[0225] When administered to an animal, a recombinant virus vaccine of the present invention infects cells within the immunized animal and directs the production of a protective protein or RNA nucleic acid molecule that is capable of protecting the animal from flea infestation as disclosed herein. For example, a recombinant virus vaccine comprising a flea HMT and/or HNC nucleic acid molecule of the present invention is administered according to a protocol that results in the animal producing a sufficient immune response to protect itself from flea infestation. A preferred single dose of a recombinant virus vaccine of the present invention is from about 1×10
[0226] A recombinant cell vaccine of the present invention includes recombinant cells of the present invention that express at least one protein of the present invention. Preferred recombinant cells for this embodiment include Salmonella,
[0227] The efficacy of a therapeutic composition of the present invention to protect an animal from flea infestation can be tested in a variety of ways including, but not limited to, detection of protective antibodies (using, for example, proteins or mimetopes of the present invention), detection of cellular immunity within the treated animal, or challenge of the treated animal with the fleas to determine whether the treated animal is resistant to infestation. Challenge studies can include direct administration of fleas to the treated animal. In one embodiment, therapeutic compositions can be tested in animal models such as mice. Such techniques are known to those skilled in the art.
[0228] One therapeutic composition of the present invention includes an inhibitor of flea HMT and/or HNC protein activity, i.e., a compound capable of substantially interfering with the function of a flea HMT and/or HNC protein susceptible to inhibition by an inhibitor of flea HMT and/or HNC protein activity. An inhibitor of HMT and/or HNC protein activity can be identified using flea HMT and/or HNC proteins of the present invention. An inhibitor of HMT and/or HNC protein function can be identified using flea HMT and/or HNC proteins of the present invention. A preferred inhibitor of HMT and/or HNC protein function is a compound capable of substantially interfering with the function of a flea HMT and/or HNC protein and which does not substantially interfere with host animal proteins. As used herein, a compound that does not substantially inhibit or interfere with host animal proteins is one that, when administered to a host animal, the host animal shows no significant adverse effects attributable to the compound and which, when administered to an animal in an effective manner, is capable of protecting that animal from flea infestation.
[0229] One embodiment of the present invention is a method to identify a compound capable of inhibiting HMT and/or HNC protein activity of a flea. Such a method includes the steps of (a) contacting (e.g., combining, mixing) an isolated flea HMT and/or HNC protein, preferably a
[0230] Putative inhibitory compounds to screen include antibodies (including fragments and mimetopes thereof), putative substrate analogs, and other, preferably small, organic or inorganic molecules. Methods to determine HMT and/or HNC protein activity are known to those skilled in the art; see, for example, the Examples section of the present application. Methods to determine binding of a putative inhibitory compound to a HMT and/or HNC protein of the present invention are known to those of skill in the art and include, for example, determining changes in molecular mass using surface plasmon resonance (e.g., determining light scatter by an inhibitor of a HMT and/or HNC protein, before and after contacting the inhibitor or protein with a HMT and/or HNC protein or inhibitor, respectively) or screening for compounds that inhibit interaction between a HMT and/or HNC protein and a substrate.
[0231] A preferred method to identify a compound capable of inhibiting HMT and/or HNC protein activity includes contacting an isolated flea HMT and/or HNC protein having an amino acid sequence selected from the group consisting of: (a) a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:154, SEQ ID NO:160, SEQ ID NO:163, SEQ ID NO:169, SEQ ID NO:1862, SEQ ID NO:1868, SEQ ID NO:1873, SEQ ID NO:1879, SEQ ID NO:1883, SEQ ID NO:1888, SEQ ID NO:1897, SEQ ID NO:1902, SEQ ID NO:1915, SEQ ID NO:1920, SEQ ID NO:1925, and/or SEQ ID NO:1930, and/or a protein encoded by a nucleic acid molecule of Table I, Table II, Table III and/or Table IV; (b) a protein comprising an at least 25 consecutive amino acid portion identical in sequence to a consecutive amino acid portion of a sequence as set forth in (a), wherein the protein has HMT and/or HNC protein activity; (c) a protein comprising a fragment of a protein as set forth in (a), wherein the fragment has an activity selected from the group consisting of binding to a HMT and/or HNC molecule and hydrolyzing a HMT and/or HNC protein substrate; and (d) a protein encoded by an allelic variant of a nucleic acid molecule that encodes any protein of (a), (b), or (c), with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein has HMT and/or HNC protein activity; and determining if the putative inhibitory compound inhibits the activity.
[0232] Another embodiment of the present invention is an assay kit to identify an inhibitor of a flea HMT and/or HNC protein of the present invention. This kit comprises an isolated flea HMT and/or HNC protein of the present invention, and a means for determining inhibition of an activity of flea HMT and/or HNC protein, where the means enables detection of inhibition. Detection of inhibition of flea HMT and/or HNC protein identifies a putative inhibitor to be an inhibitor of flea HMT and/or HNC protein. Means for determining inhibition of flea HMT and/or HNC protein include an assay system that detects binding of a putative inhibitor to a flea HMT and/or HNC molecule, and an assay system that detects interference by a putative inhibitor of the ability of flea HMT and/or HNC protein to hydrolyze a substrate. Means and methods are described herein and are known to those skilled in the art.
[0233] The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention. The following examples include a number of recombinant DNA and protein chemistry techniques known to those skilled in the art; see, for example, Sambrook et al., ibid.
EXAMPLE 1
[0234] This Example describes the isolation of RNA from the hindgut and Malpighian tubules (HMT) of
[0235] Approximately 10,000 hindguts and Malpighian tubules were dissected from equal numbers of cat blood fed and unfed adult
[0236] Poly-A enriched mRNA was used to construct a cDNA library using subtractive hybridization and suppression PCR as follows. Subtractive hybridization and suppression PCR was conducted using a PCR-Select™ cDNA Subtraction Kit, available from Clontech Laboratories, Inc., Palo Alto, Calif. according to the manufacturer's instructions. Briefly, this kit uses subtractive hybridization and suppression PCR to specifically amplify cDNA sequences that are present in the tester cDNA and absent in the driver cDNA, thus enriching for tester-specific sequences. The efficiency of the subtraction process can be assessed by semi-quantitative PCR and by comparing the ethidium bromide staining patterns of the subtracted and unsubtracted samples on agarose gels as described in section V.D. of the manufacturer's protocol. For the semi-quantitative PCR, three genes with mRNAs known to be expressed outside of the HMT tissue were used to test for specific subtraction. These genes encoded putative actin, N-aminopeptidase, and serine protease proteins.
[0237] Subtractive hybridization and suppression PCR was conducted under the following conditions. Two micrograms (μg) of HMT mRNA was used as the template for synthesis of the tester material and 2 μg of non-HMT mRNA was used as template for synthesis of the driver material in this reaction. The number of cycles used in the selective amplification steps was optimized using the manufacturer's protocols. Optimization resulted in the use of 24 rather than the standard 27 cycles of primary PCR in combination with 15 cycles of secondary PCR rather than the standard 12 cycles.
[0238] The products from the suppressive PCR reaction were ligated into the pCR®2.1 vector, available from Invitrogen, Carlsbad, Calif., using an Original TA Cloning® Kit, available from Invitrogen. The ligation reaction was then used to transform INVαF′ One Shot™ competent cells, available from Invitrogen, which were plated on Luria broth (LB) agar with 50 micrograms per milliliter (“g/ml) ampicillin, available from Sigma-Aldrich Co., St. Louis, MO, and 50 “g/ml 5-bromo-4-chloro-3-indoyl β-D-galactopyranoside (X-Gal), available from Fisher Biotech, Fair Lawn, N.J. Transformed colonies were amplified and the DNA isolated using the standard alkaline lysis procedure described by Sambrook et al., ibid.
[0239] Automated cycle sequencing of DNA samples was performed using an ABI PRISM™ Model 377, available from Perkins Elmer, with XL upgrade DNA Sequencer, available from PE Applied Biosystems, Foster City, Calif., after reactions were carried out using the PRISM™ Dye Terminator Cycle Sequencing Ready Reaction Kit or the PRISM™ dRhodamine Terminator Cycle Sequencing Ready Reaction Kit or the PRISM™ BigDye™ Terminator Cycle sequencing Ready Reaction Kit, available from PE Applied Biosystems, following the manufacturer's protocol, hereinafter “standard sequencing methods”. Sequence analysis was performed using the MacVector™ sequence analysis software, available from International Biotechnologies Inc., New Haven, Conn., and the Wisconsin Package Version 9.0 sequence analysis software, available from Genetics Computer Group (GCG), Madison, Wis., hereinafter referred to as GCG version 9.0, using default parameters. Each sequence read was trimmed of vector sequence at either end and submitted for a search through the National Center for Biotechnology Information (NCBI), National Library of Medicine, National Institute of Health, Baltimore, Md., using the BLAST network. This database includes SwissProt+PIR+SPupdate+GenPept+GPUpdate+PDB databases. The search was conducted using the xBLAST function, which compares the translated sequences in all 6 reading frames to the protein sequences contained in the database. Clones with significant homology to sequences in the GenBank database were grouped according to proposed function and are listed in Table II. Clones with no significant homology to sequences in the GenBank database were searched manually for open reading frames and are listed in Table IV.
[0240] An unsubtracted HMT cDNA library was constructed as follows. Approximately 10,000 HMT tissues were dissected from equal numbers of unfed and cat blood-fed adult
EXAMPLE 2
[0241] This Example describes the isolation of RNA from the head and nerve cord (HNC) of
[0242] Approximately 4,000 heads and attached nerve cords, including the terminal abdominal ganglia were dissected from equal numbers of cat blood-fed and unfed adult
[0243] Suppression subtractive PCR was conducted as described in Example 1 using a PCR-Select™ cDNA Subtraction kit, available from Clontech, under the following conditions. Two micrograms (μg) of HNC mRNA was used as the template for synthesis of the tester material and 2 μg of non-HMT mRNA was used as template for synthesis of the driver material in this reaction. The number of cycles used in the selective amplification steps was optimized using the manufacturer's protocols. Optimization resulted in the use of 24 rather than the standard 27 cycles of primary PCR in combination with either 12 or 15 cycles of secondary PCR. cDNA pools from various PCR cycling combinations were ligated into the TA vector using a TA cloning kit, available from Invitrogen. Aliquots of ligation reaction were transformed into Ultramax DH5∝™ bacteria, available from Gibco-BRL, Gaithersburg, MD. Portions of the transformation mixes were used to inoculate LB broth cultures containing 100 μg/ml of ampicillin. The overnight cultures were plated to generate discreet colonies which were used individually for overnight cultures for plasmid preps. Transformed colonies were amplified and the DNA isolated using the standard alkaline lysis procedure described by Sambrook et al., ibid.
[0244] Automated cycle sequencing of DNA samples was performed using the standard sequencing methods described in Example 1. Sequence analysis was performed using the MacVector™ sequence analysis software, available from International Biotechnologies Inc., New Haven, Conn., and the Wisconsin Package Version 9.0 sequence analysis software, available from Genetics Computer Group (GCG), Madison, Wis., hereinafter referred to as GCG version 9.0, using default parameters. Each sequence read was trimmed of vector sequence at either end and submitted for a xBLAST search as described in Example 1. Clones with significant homology to sequences in the GenBank database were grouped according to proposed function and are listed in Table I. Clones with no significant homology to sequences in the GenBank database were searched manually for open reading frames and are listed in Table III.
[0245] An unsubtracted cDNA library was constructed as follows. Approximately 6400 head and nerve cords were dissected from
EXAMPLE 3
[0246] This example describes the production of a
[0247] Total RNA was extracted from adult fed and unfed fleas as follows. Approximately 1000 adult fed fleas and 1000 adult unfed fleas were frozen on dry ice and separately ground into powder using a mortar and pestle and total RNA was extracted from each powder as follows. Ten ml of solution D (4 M guanidine isothiocyanate, 25 mM Sodium Citrate pH 7.0, 1.5% Sarcosyl, 0.5 M 2-mercaptoethanol) were added to the powder and the suspension was mixed by shaking. One ml of 2M sodium acetate, pH 4.0 and 3 ml of pH 4.7 phenol/chloroform/isoamyl alcohol (125:24:1), available from Sigma, were added and the suspension was mixed on a vortex shaker then incubated on ice for 15 minutes. Following incubation, the mixture was centrifuged at 10,000× g for 20 minutes and the supernatant was removed and extracted twice with pH 4.7 phenol/chloroform/isoamyl alcohol. Next, an equal volume of isopropanol was added to the supernatant and incubated at −20° C. for 2 hours followed by centrifugation at 10,000× g for 20 minutes. Following centrifugation, the supernatant was removed and discarded and the pellet was washed in 70% ethanol and allowed to dry at room temperature. The pellet was resuspended in 10 mM Tris 1 mM EDTA pH 8.0. Spectrophotometer analysis indicated that the yield of total RNA from unfed fleas was 1140 μg and the yield from fed fleas was 1500 μg.
[0248] Six-hundred μg from each of the fed and unfed adult flea total RNA extractions were combined and mRNA was then extracted using a mRNA Purification Kit, available from Amersham Pharmacia Biotech, Piscataway, N.J., using the manufacture's protocol. Approximately 15-25 μg of mRNA were isolated based on spectrophotometer analysis and ethidium bromide staining. One μg of purified mRNA was used as template to construct a RACE cDNA pool using a Marathon cDNA Amplification Kit, available from Clontech Laboratories, Inc., Palo Alto, Calif., according to the manufacture's instructions.
EXAMPLE 4
[0249] This example describes the cloning, sequencing, recombinant protein expression and purification of a
[0250] A TA clone from the HMT EST library described in Example 1 was sequenced using standard sequencing methods and shown to have significant homology to allantoinase genes. This clone was digested with EcoRI to excise an insert 682 nucleotides in length, referred to as flea nucleic acid molecule nCfALN
[0251] The
[0252] Translation of SEQ ID NO:1 suggests that nucleic acid molecule nCfALN
[0253] Comparison of amino acid sequence SEQ ID NO:2 with amino acid sequences reported in GenBank indicates that SEQ ID NO:2 showed the most homology, i.e., about 48.6% identity, with a
[0254] The coding region of nCfALN
[0255] The recombinant cell was grown under standard conditions and then incubated in the presence of 0.5 μM isopropylthio-β-galactoside (IPTG) to induce expression of recombinant protein, predicted to be approximately 42.2 kDa. Expression was confirmed using Coomassie-blue-stained Tris-glycine gel and by Western blot using a T7 tag antibody, available from Novagen, which showed expression of an about 55-kDa protein. The protein product was purified by liquid chromatography using a HiTrap™ chelating column charged with NiCl
[0256] A Northern Blot analysis was conducted as follows to determine whether allantoinase is expressed exclusively in HMT tissues. HMT tissues were dissected from 1000 adult cat blood-fed
[0257] Approximately 10 μg of each RNA was added to separate tubes containing 18.75 μl of loading buffer, which consists of 50% formamide, 16% formaldehyde, 17% water, 7% glycerol, 1× MOPS buffer (a 1:20 dilution of 0.4 M 93-[N-morpholino]propanesulfonic acid (MOPS), 0.1 M sodium acetate, and 20 MM EDTA), 10 μl ethidium bromide, and 10 μl bromophenol blue dye, all available from Sigma. The tubes were heated to 95° C. for 2 minutes then placed on ice. The RNA samples were separated by gel electrophoresis on a 1.5% agarose gel with 3.2% formaldehyde and 1× MOPS buffer; the gel was then soaked in water for 30 minutes prior to transfer to remove excess formaldehyde. The gel was then transferred using standard techniques, described by Sambrook et al., ibid, with 10× SSPE as the transfer buffer onto Nytran® nylon membrane, available from Schleicher and Schuell Inc., Keene, N.H. The membrane was UV cross-linked using the Stratalinker®, available from Stratagene, then prehybridized at 42° C. in 50% formamide, 5× SSPE, 1.2% SDS, 5× Denhardt's reagent, 2.5 mM EDTA, and 100 μg/ml salmon sperm DNA. A probe comprising the allantoinase EST nucleic acid molecule, nCfALN
[0258] Northern Blot analysis was also conducted to determine whether allantoinase mRNA is expressed only in certain stages of the flea life cycle and whether allantoinase mRNA expression is influenced by feeding. Total RNA was extracted as described above from 1000 fleas at each of the following flea life stages; eggs, first instar larvae, third instar larvae, wandering larvae and pupae and from 1000 adult fleas under the following feeding conditions; unfed, fed on cat blood for 15 minutes, fed on cat blood for 2 hours, fed on cat blood for 8 hours, and fed on cat blood for 24 hours. Each RNA sample was separated by gel electrophoresis, transferred to nylon membrane and hybridized with α-
EXAMPLE 5
[0259] This example describes the cloning, sequencing, recombinant protein expression and purification of a
[0260] A TA clone from the HMT EST library described in Example 1 was sequenced using standard sequencing methods and shown to have homology to a chitinase-like gene from
[0261] The
[0262] Translation of SEQ ID NO:7 suggests that nucleic acid molecule nCfCBP
[0263] Comparison of amino acid sequence SEQ ID NO:8 with amino acid sequences reported in GenBank indicates that SEQ ID NO:8 showed the most homology, i.e., about 26% identity with a
[0264] A nucleic acid molecule comprising nucleotides 59 through 827 of SEQ ID NO:7, encoding a predicted mature flea chitin-binding protein, was PCR amplified from the pBluescript™ clone described above as the template, using sense primer CBP-FE, having nucleotide sequence 5′ CGG GAT CCT GCT GAC AGG AAT TCG CCC AC 3′, having a BamHI site indicated in bold, designated herein as SEQ ID NO:39, and anti-sense primer CBP-RE, having nucleotide sequence 5′ CAT GGT ACC CCT GGT TTA AGC CTT ACT TAG C 3′, having a KpnI site indicated in bold, designated herein as SEQ ID NO:38 PCR reactions were performed using standard PCR reaction and thermocycling conditions described in Example 4. The PCR product was digested with BamHI and KpnI and ligated into the vector pTrcHisB, available from Invitrogen, that had been digested with BamHI and KpnI and treated with alkaline phosphatase. The resulting recombinant molecule, referred to herein as pTrc-nCfCBP
[0265] Northern Blot analysis was conducted as described in Example 4 to determine whether CBP mRNA is expressed in only HMT tissue, only in certain stages of the flea life cycle and whether CBP mRNA expression is influenced by feeding. Total RNA was extracted from flea tissues, life stages and feeding conditions as described in Example 4. Each RNA sample was separated by gel electrophoresis, transferred to a nylon membrane and hybridized with α-
EXAMPLE 6
[0266] This example describes the cloning and sequencing of a
[0267] A TA clone from the HMT EST library described in Example 1 was sequenced using standard sequencing methods and shown to have homology to the nervous system antigen 1 gene from
[0268] The
[0269] Translation of SEQ ID NO:13 suggests that nucleic acid molecule nCfNKAB
[0270] Comparison of amino acid sequence SEQ ID NO:14 with amino acid sequences reported in GenBank indicates that SEQ ID NO:14 showed the most homology, i.e., about 46% identity, with a
EXAMPLE 7
[0271] This example describes the cloning and sequencing of a
[0272] A TA clone from the HMT EST library described in Example 1 was sequenced using standard sequencing methods and shown to have homology to a human ligand-gated chloride channel nucleic acid molecule. The clone was digested with EcoRI to excise an insert about 376 nucleotides in length, referred to as flea LGIC nucleic acid molecule nCfLGIC
[0273] The
[0274] Translation of SEQ ID NO:19 suggests that nucleic acid molecule nCfLGIC
[0275] Comparison of amino acid sequence SEQ ID NO:20 with amino acid sequences reported in GenBank indicates that SEQ ID NO:20 showed the most homology, i.e., about 23% identity, with a
[0276] Northern Blot analysis was conducted as described in Example 4 to determine whether LGIC mRNA is expressed in only HMT tissue. Total RNA was extracted from HMT tissues and non-HMT tissues as described in Example 4. Each RNA sample was separated by gel electrophoresis, transferred to nylon membranes and hybridized with α-
[0277] Additional nucleic acid sequence corresponding to the coding regions at the 5′ end of the LGIC cDNA described above was isolated by PCR using the RACE cDNA pool prepared as described in Example 3 as the template. A first PCR reaction was conducted using reverse primer LGIC-R4, which is complementary to nucleotides 200-223 of SEQ ID NO:19, having a nucleic acid sequence 5′ GCG ATA CTG GTG GTA CTG GTG AAG 3′, denoted herein as SEQ ID NO:1932 was used with the forward linker primer Adapter Primer 1, having a nucleic acid sequence 5′ CCA TCC TAA TAC GAC TCA CTA TAG GGC 3′, denoted herein as SEQ ID NO:1933 using standard PCR reaction conditions and the following thermocycling conditions: (1) 94° C. for 30 seconds, (2) 5 cycles of 94° C. for 10 seconds then 72° C. for 4 minutes, (3) 5 cycles of 94° C. for 10 seconds then 70° C. for 4 minutes, and (4) 25 cycles of 94° C. for 10 seconds then 68° C. for 4 minutes. The reaction product was separated on a 1.5% agarose gel and stained by ethidium bromide, but no clear bands were seen. The first PCR reaction product was diluted 1:50 in water and used as template for a second PCR reaction using reverse primer LGIC-R5, which is complementary to nucleotides 88-110 of SEQ ID NO:19, having a nucleic acid sequence 5′ GAG GTG GTT GTC TTC AGT GGT TG 3′, denoted herein as SEQ ID NO:1934 and forward Adapter Primer 2, having a nucleic acid sequence 5′ ACT CAC TAT AGG GCT CGA GCG GC 3′, denoted herein as SEQ ID NO:1935 under the same reaction conditions described for the first PCR reaction. The reaction product was separated by electrophoresis on a 1.5% agarose gel and stained with ethidium bromide revealing an approximately 700 bp band. This band was cut from the gel and purified using the QIAquick Gel Extraction Kit, then ligated into the pCR II TA Cloning vector, available from Invitrogen Corporation, Carlsbad, Calif., using the manufacture's protocol. This clone, referred to herein as nCfLGIC
[0278] Comparison of amino acid sequence SEQ ID NO:1862 with amino acid sequences reported in GenBank indicates that SEQ ID NO:1862 showed the most homology, i.e., 31.4% identity with glycine receptor Alpha 3 chain precursor cDNA from
[0279] A LGIC nucleic acid molecule for recombinant expression of the predicted extracellular domain was produced as follows. In order to ligate the region encoding the predicted extracellular domain of the LGIC cDNA into the InsectSelect™ expression vector pIB/V5-His, two separate but overlapping DNA fragments were generated to be used as the template in the PCR overlap extension. To generate a 3′ DNA fragment, a first PCR reaction was conducted using forward primer LGIC-ECD-D2F, which corresponds to nucleotides 2-25 of SEQ ID NO:19, having a nucleic acid sequence 5′ CAA TTT TAA ACG CAT CCA CGA CCG 3′, denoted herein as SEQ ID NO:1936, and reverse primer LGIC-ECD-RE, which is complementary to nucleotides 937-961of SEQ ID NO:19, having a nucleic acid sequence 5′ CCG CTC GAG CGA CCC ATT TCA CGA CTT ATT TGA ATC G 3′, denoted herein as SEQ ID NO:1937 and having a XhoI site indicated in bold, to amplify nucleotides 2-963 from SEQ ID NO:19 which was used as template under standard PCR reaction conditions and the following thermocycling conditions: (1) 94° C. for 30 seconds, (2) 25 cycles of 94° C. for 10 seconds, 55° C. for 10 seconds, and 72° C. for 3 minutes. The products of this reaction were separated on a 1.5% agarose gel, and a band corresponding to an approximately 960 nucleotide molecule was cut from the gel and purified using the QIAquick Gel Extraction Kit as described above. To generate a 5′ cDNA fragment, a second PCR reaction was conducted using reverse primer LGIC-R5 (SEQ ID NO:1934) and forward primer LGIC-ECD-FE, which corresponds to nucleotides 188-215 of SEQ ID NO:1859, having a nucleic acid sequence 5′ GGA ATT CTA AAA TGC ACA ACA AAA TCC TGG TCC TGG 3′, denoted herein as SEQ ID NO:1938, and having an EcoRI site indicated in bold, using SEQ ID NO:1859 as the template under the thermocycling conditions described for generating the 3′ fragment. The products of this reaction were separated on a 1.5% agarose gel, and a band corresponding to an approximately 425 nucleotide molecule was cut from the gel and purified using the QIAquick Gel Extraction Kit as described above.
[0280] For the PCR overlap extension reaction, the 5′ and 3′ cDNA fragments described above were used as the template in a PCR reaction with forward primer LGIC-ECD-FE and reverse primer LGIC-ECD-RE under the thermocycling conditions described for generating the 5′ and 3′ fragments. The products of this reaction were separated on a 1.5% agarose gel, and a band corresponding to an approximately 1300 nucleotide molecule, as visualized by agarose gel electrophoresis and ethidium bromide staining, referred to herein as nCfLGIC
[0281] The product of the PCR overlap extension reaction was the digested with EcoRI and XhoI restriction endonucleases, available from New England BioLabs, Inc., Beverly, Mass., for 18 hours at 37°. The digestion product was purified using the QIAquick Nucleotide Removal Kit, available from Qiagen, and ligated into the vector pIB/V5-His which had also been digested with EcoRI and XhoI and treated with shrimp alkaline phosphatase, available from New England BioLabs, Inc. for 30 minutes at 37. Following standard transformation procedures, a bacterial clone containing the plasmid pIB/V5-His-nCfLGIC
EXAMPLE 8
[0282] This example describes the cloning and sequencing of a
[0283] A TA clone from the HMT EST library described in Example 1 was sequenced using standard sequencing methods and shown to have homology to an ANON/23DA gene from
[0284] The
[0285] Translation of SEQ ID NO:25 suggests that nucleic acid molecule nCfANON
[0286] Comparison of amino acid sequence SEQ ID NO:26 with amino acid sequences reported in GenBank indicates that SEQ ID NO:26 showed the most homology, i.e., about 65% identity, with a
[0287] Northern Blot analysis was conducted as described in Example 4 to determine whether ANON mRNA is expressed in only HMT tissue, only in certain stages of the flea life cycle and whether ANON mRNA expression is influenced by feeding. Total RNA was extracted from flea tissues, life stages and feeding conditions as described in Example 4. Each RNA sample was separated by gel electrophoresis, transferred to nylon membranes and hybridized with α-
EXAMPLE 9
[0288] This example describes the cloning and sequencing of a
[0289] A TA clone from the HMT EST library described in Example 1 was digested with EcoRI to excise an insert about 432 nucleotides in length, referred to as nCfMALV
[0290] Sequence information from nCfMALV
[0291] The second PCR reaction resulted in an approximately 1000 bp PCR product which was separated by electrophoresis on a 1.5% agarose gel, excised and purified using a Gel Purification Kit, available from Qiagen. The purified PCR product was ligated into the pCRII™, Original TA cloning vector, available from Invitrogen. The ligation reaction was then used to transform INVαF′ One Shot™ competent cells, available from Invitrogen, which were plated on LB agar with 50 micrograms per milliliter (μg/ml) ampicillin, available from Sigma-Aldrich Co., and 50 μg/ml X-Gal, available from Fisher Biotech. A clone was isolated from the ligation mix containing a nucleic acid molecule of about 765 base pairs, referred to herein as nCfMALV
[0292] Translation of SEQ ID NO:31 suggests that nucleic acid molecule nCfMALV
[0293] Comparison of amino acid sequence SEQ ID NO:32 with amino acid sequences reported in GenBank indicates that SEQ ID NO:32 showed the most homology, i.e., about 71% identity, with a
EXAMPLE 10
[0294] This example describes the cloning, sequencing, and recombinant expression of a
[0295] A
[0296] To isolate a flea OS-D nucleic acid molecule encoding a full-length OS-D protein, nucleic acid molecule nCfOSD
[0297] A DNA fragment of about 365 nucleotides, referred to herein as nCfOSD
[0298] Translation of SEQ ID NO:37 suggests that nucleic acid molecule nCfOSD
[0299] Comparison of amino acid sequence SEQ ID NO:38 with amino acid sequences reported in GenBank indicates that SEQ ID NO:38 showed the most homology, i.e., about 60% identity, with a
[0300] A nucleic acid molecule comprising nucleotides 91 through 447 of SEQ ID NO:37, encoding a predicted mature flea OS-D protein, was PCR amplified using the pBluescript™ clone described above as the template, using sense primer OSD-FE, having nucleotide sequence 5′ CGC GGA TCC AGA AGA TAA ATA TAC TAG CAA ATT TGA TAA C 3′, having a BamHI site indicated in bold, designated herein as SEQ ID NO:61, and anti-sense primer OSD-RE, having nucleotide sequence 5′ GAG GAA TTC CTC TTT TTG GAA ATT TAA ACT GTA ACG G 3′, having an EcoRI site indicated in bold, designated herein as SEQ ID NO:62. PCR reactions were performed using standard PCR reaction and thermocycling conditions described in Example 4; the product was separated by agarose gel electrophoresis, and a fragment was excised and purified using a Gel Purification Kit, available from Qiagen. The fragment was digested with BamHI and EcoRI and ligated into the vector pTrcHisB, available from Invitrogen, that had been digested with BamHI and EcoRI and treated with alkaline phosphatase. The resulting recombinant molecule, referred to herein as pTrc-nCfOSD
[0301] The recombinant cell was grown under standard conditions then incubated in the presence of 0.5 mM IPTG to induce expression of recombinant protein, predicted to be approximately 17-kDa. Expression of protein was confirmed using Coomassie-blue-stained Tris-glycine gel and by Western blot using a T7 tag antibody which showed expression of an about 17 kDa protein.
[0302] A Northern Blot analysis was conducted as follows to determine whether OS-D mRNA is expressed exclusively in HNC tissues. HNC tissues were dissected from 1500 adult cat blood-fed
[0303] Approximately 15 μg of each RNA were separated by electrophoresis on either Glyoxal gels with RNA prepared according to Burnett, Biotechniques, 22:4, pp. 668-671, 1997, or formaldehyde gels with RNA prepared according to Sambrook et al., ibid. Following electrophoresis, RNA was blotted to Hybond N nylon membranes, available from Amersham, according to the protocols described in Burnett and Sambrook et el. ibid.