U.S. patent application number 09/991936 was filed with the patent office on 2003-04-17 for flea head, nerve cord, hindgut and malpighian tubule nucleic acid molecules, proteins and uses thereof.
Invention is credited to Brandt, Kevin S., Gaines, Patrick J., Stinchcomb, Dan T., Wisnewski, Nancy.
Application Number | 20030073827 09/991936 |
Document ID | / |
Family ID | 22436573 |
Filed Date | 2003-04-17 |
United States Patent
Application |
20030073827 |
Kind Code |
A1 |
Brandt, Kevin S. ; et
al. |
April 17, 2003 |
Flea head, nerve cord, hindgut and malpighian tubule nucleic acid
molecules, proteins and uses thereof
Abstract
The present invention relates to flea head, nerve cord, hindgut
and Malpighian tubule proteins; to flea head, nerve cord, hindgut
and Malpighian tubule nucleic acid molecules, including those that
encode such flea head, nerve cord, hindgut and Malpighian tubule
proteins; to antibodies raised against such flea head, nerve cord,
hindgut and Malpighian tubule proteins; and to compounds that
inhibit flea head, nerve cord, hindgut and Malpighian tubule
protein activity. The present invention also includes methods to
obtain such proteins, nucleic acid molecules, antibodies, and
inhibitory compounds. Also included in the present invention are
therapeutic compositions comprising proteins, nucleic acid
molecules, or protective compounds derived from proteins of the
present invention as well as the use of such therapeutic
compositions to protect animals from flea infestation. Also
included in the present invention is the use of flea head, nerve
cord, hindgut and Malpighian tubule proteins to derive inhibitory
compounds.
Inventors: |
Brandt, Kevin S.; (Windsor,
CO) ; Gaines, Patrick J.; (Fort Collins, CO) ;
Stinchcomb, Dan T.; (Fort Collins, CO) ; Wisnewski,
Nancy; (Fort Collins, CO) |
Correspondence
Address: |
HESKA CORPORATION
INTELLECTUAL PROPERTY DEPT.
1613 PROSPECT PARKWAY
FORT COLLINS
CO
80525
US
|
Family ID: |
22436573 |
Appl. No.: |
09/991936 |
Filed: |
November 21, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09991936 |
Nov 21, 2001 |
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09543668 |
Apr 7, 2000 |
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60128704 |
Apr 9, 1999 |
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Current U.S.
Class: |
536/23.1 ;
435/183; 435/320.1; 435/325; 435/6.15; 435/69.1 |
Current CPC
Class: |
C12N 2799/021 20130101;
A61K 2039/505 20130101; A61K 38/00 20130101; A61P 33/14 20180101;
A61P 17/00 20180101; A61K 48/00 20130101; A61K 39/00 20130101; C07K
14/4359 20130101; A61P 37/08 20180101 |
Class at
Publication: |
536/23.1 ; 435/6;
435/69.1; 435/320.1; 435/325; 435/183 |
International
Class: |
C07H 021/04; C12Q
001/68; C12N 009/00; C12P 021/02; C12N 005/06 |
Claims
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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (2) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (2) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (2) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about 37.degree.
C. and (2) washing in a solution comprising 1.times. SSC and 0%
formamide, at a temperature of about 47.5.degree. 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.
Description
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 Bombyx mori (GenBank accession #1841851)
is reported to have weak similarity with the chitin-binding domain
of insect chitinases; however, it has no significant similarity to
the catalytic regions of known chitinases, and therefore is not
expected to have chitinase activity. The chitinase-like protein of
B. mori is also similar to the peritrophin family of proteins
located in the peritrophic matrix of insects. These proteins
contain putative chitin-binding domains but have no other apparent
homology to any known proteins. Without being bound by theory, it
is believed that these proteins bind chitin and are a structural
component of the peritrophic matrix. As such, flea chitin binding
protein represents a novel target for anti-flea vaccines and
chemotherapeutic drugs.
[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 (.alpha.)
subunit, a 40-kDa beta (.beta.) subunit, and a 6-kDa gamma
(.gamma.) subunit. Most insects express three isotypes of the
.beta. subunit, each being expressed in a tissue and cell-type
dependent manner. The .alpha. subunit has 8 transmembrane domains
whereas the .beta. and .gamma. subunits have just one. The .alpha.
subunit mediates ATPase and ion transporting activities and
together with the .gamma. subunit comprises the site for cardiac
glycoside (ouabain) binding. The .beta. 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 Drosophila melanogaster
antenna cDNA library encodes a protein that shares features common
to vertebrate odorant-binding proteins, but has a primary structure
unlike odorant-binding proteins. The encoded protein is also
homologous to a family of soluble chemosensory proteins from the
chemosensory organ of the desert locust, Schistocerca gregaria. As
such, a flea OS-D protein of the present invention represents a
novel target for anti-flea vaccines and chemotherapeutic drugs.
[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 Drosophila melanogaster, and approximately
the same identity with CSRLLBP, chemical sense related lipophilic
ligand binding protein of Phormia regina). Without being bound by
theory, it is believed that these proteins are involved in the
perception of odors or pheromones, such as the ability to sense the
presence of a host or mate. As such, a flea CLBP protein of the
present invention represents a novel target for anti-flea vaccines
and chemotherapeutic drugs.
[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, Lucilia cuprina. It has been shown in this insect
that ingestion of antibodies against peritrophins inhibits the
growth of larvae and can result in increased larval mortality. It
has also been shown that the ingestion of antibodies against
peritrophins reduces the permeability of the peritrophic matrix in
L. cuprina larvae. This in turn may inhibit the movement of
digested food across the peritrophic matrix to the gut epithelium,
resulting in starvation. As such, a flea peritrophin of the present
invention represents a novel target for anti-flea vaccines and
chemotherapeutic drugs.
[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. C.
felis AUP shares some homology to Drosophila melanogaster
anoxia-regulated gene product fau. The Drosophila melanogaster fau
gene has no homology to previously described database entries, but
localizes to laminal and cortical neurons of the Drosophila CNS by
in situ hybridization, and plays and important role in response to
O2 deprivation as measured by impaired recovery time of transgenic
flies over-expressing fau to anoxia. As such, a flea AUP of the
present invention represents a novel target for anti-flea vaccines
and chemotherapeutic drugs.
[0032] A flea 7B2 protein has some BLAST homology to the
neuroendocrine protein 7B2 from various organisms, including
Drosophila, C. elegans, the pond snail Lymnaea stagnalis, and
humans. 7B2 has been implicated in activation of prohormone
convertase 2 (PC2) an important neuroendocrine precursor processing
endoprotease. Additionally, 7B2 was found to be critical in islet
hormone processing in mice using null mutants which displayed
hypoglycemia, hyperproinsulinemia and hypoglucagonemia. As such, a
flea 7B2 of the present invention represents a novel target for
anti-flea vaccines and chemotherapeutic drugs.
[0033] Flea allantoinase nucleic acid molecules of known length
isolated from C. felis are denoted "nCfALN.sub.#", for example
nCfALN.sub.2057, wherein "#"refers to the number of nucleotides in
that molecule, and allantoinase proteins of known length are
denoted "PCfALN.sub.#" (for example PCfALN.sub.384) wherein
"#"refers to the number of amino acid residues in that molecule.
Similarly, C. felis CBP nucleic acid molecules and proteins of
known length are denoted "nCfCBP.sub.#" and "PCfCBP.sub.#",
respectively; C. felis NKAB nucleic acid molecules and proteins of
known length are denoted "nCfNKAB.sub.#"and "PCfNKAB.sub.#",
respectively; C. felis LGIC nucleic acid molecules and proteins of
known length are denoted "nCfLGIC.sub.#" and "PCfLGIC.sub.#",
respectively; C. felis ANON nucleic acid molecules and proteins of
known length are denoted "nCfANON.sub.#" and "PCfANON.sub.#",
respectively; C. felis MALV nucleic acid molecules and proteins of
known length are denoted "nCfMALV.sub.#" and
"PCfALV.sub.#"respectively; C. felis OS-D nucleic acid molecules
and proteins of known length are denoted "nCfOSD#"and
"PCfOSD.sub.#respectively; C. felis NMDA nucleic acid molecules and
proteins of known length are denoted "nCNMDA" and
"PCfNMDA.sub.#respectiv- ely; C. felis CLBP nucleic acid molecules
and proteins of known length are denoted "nCfCLBP.sub.#" and
"PCfCLBP.sub..TM. respectively, C. felis NAH nucleic acid molecules
and proteins of known length are denoted "nCfNAH.sub.#"and
"PCfNAH.sub.# respectively, C. felis CLIC nucleic acid molecules
and proteins of known length are denoted "nCfCLIC.sub.#" and
"PCfCLIC.sub.# respectively, C. felis PL2 nucleic acid molecules
and proteins of known length are denoted "nCfPL2.sub..TM.," and
"PCfPL2.sub.# respectively, C. felis PL3 nucleic acid molecules and
proteins of known length are denoted "nCfPL3.sub.# " and
"PCfPL3.sub.# respectively, C. felis PL4 nucleic acid molecules and
proteins of known length are denoted "nCfPL4.sub.#" and
"PCfPL4.sub.# respectively, C. felis SVP nucleic acid molecules and
proteins of known length are denoted "nCfSVP.sub.#" and
"PCfSVP.sub.# respectively, C. felis VGCC nucleic acid molecules
and proteins of known length are denoted "nCfVGCC.sub.#" and
"PCfVGCC.sub.# respectively, C. felis AUP nucleic acid molecules
and proteins of known length are denoted "nCAUP f.sub.#" and
"PCfAUP.sub.# respectively, and C. felis 7B2 nucleic acid molecules
and proteins of known length are denoted "nCf7B2.sub.#" and
"PCf7B2.sub.# respectively.
[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 Ctenocephalides felis HMT and HNC genes. As
used herein, flea HMT and HNC genes include all regions such as
regulatory regions that control production of flea HMT and HNC
proteins encoded by such genes (such as, but not limited to,
transcription, translation or post-translation control regions) as
well as the coding region itself, and any introns or non-translated
coding regions. As used herein, a nucleic acid molecule that
"includes" or "comprises" a sequence may include that sequence in
one contiguous array, or may include the sequence as fragmented
exons such as is often found for a flea gene. As used herein, the
term "coding region" refers to a continuous linear array of
nucleotides that translates into a protein. A full-length coding
region is that coding region that is translated into a full-length,
i.e., a complete protein as would be initially translated in its
natural millieu, prior to any post-translational modifications.
[0044] One embodiment of the present invention is a C. felis ALN
gene that includes the nucleic acid sequence SEQ ID NO:1 and/or SEQ
ID NO:4, a C. felis CBP gene that includes the nucleic acid
sequence SEQ ID NO:7 and/or SEQ ID NO:10, a C. felis NKAB gene that
includes the nucleic acid sequence SEQ ID NO:13 and/or SEQ ID
NO:16, a C. felis LGIC gene that includes the nucleic acid sequence
SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:1861, and/or SEQ ID NO:1864,
a C. felis ANON gene that includes the nucleic acid sequence SEQ ID
NO:25 and/or SEQ ID NO:28, a C. felis MALV gene that includes the
nucleic acid sequence SEQ ID NO:31 and/or SEQ ID NO:34, a C. felis
OS-D gene that includes the nucleic acid sequence SEQ ID NO:37
and/or SEQ ID NO:40, a C. felis NMDA gene that includes the nucleic
acid sequence SEQ ID NO:43 and/or SEQ ID NO:46, a C. felis CLBP
gene that includes 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, a C. felis NAH gene that includes the nucleic acid sequence
SEQ ID NO:1867 and/or SEQ ID NO:1870, a C. felis CLIC gene that
includes the nucleic acid sequence SEQ ID NO:1872 and/or SEQ ID
NO:1875, a C. felis PL2 gene that includes 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, a C. felis PL3 gene that includes
the nucleic acid sequence SEQ ID NO:1887 and/or SEQ ID NO:1890, a
C. felis PL4 gene that includes the nucleic acid sequence SEQ ID
NO:1896 and/or SEQ ID NO:1899, a C. felis SVP gene that includes
the nucleic acid sequence SEQ ID NO:1901 and/or SEQ ID NO:1904, a
C. felis VGCC gene that includes the nucleic acid sequence SEQ ID
NO:1914 and/or SEQ ID NO:1917, a C. felis AUP gene that includes
the nucleic acid sequence SEQ ID NO:1919 and/or SEQ ID NO:1922, a
C. felis 7B2 gene that includes the nucleic acid sequence SEQ ID
NO:1924 and/or SEQ ID NO:1927, a C. felis gene that includes a
nucleic acid sequence of Table L Table II, Table III and/or Table
IV; as well as the complements of any of these nucleic acid
sequences. These nucleic acid sequences are further described
herein. For example, nucleic acid sequence SEQ ID NO:1 represents
the deduced sequence of the coding strand of a C. felis cDNA
denoted herein as C. felis ALN nucleic acid molecule
nCfALN.sub.2057, the production of which is disclosed in the
Examples. Nucleic acid molecule SEQ ID NO:1 comprises an apparently
full-length coding region. The complement of SEQ ID NO:1
(represented herein by SEQ ID NO:3) refers to the nucleic acid
sequence of the strand fully complementary to the strand having SEQ
ID NO:1, which can easily be determined by those skilled in the
art. Likewise, a nucleic acid sequence complement of any nucleic
acid sequence of the present invention refers to the nucleic acid
sequence of the nucleic acid strand that is fully complementary to
(i.e., can form a complete double helix with) the strand for which
the sequence is cited. For example, the complements of SEQ ID
NOs:4, 7, 10, 13, 16, 19, 22, 25, 28, 31, 34, 37, 40, 43, 46, 153,
156, 159, 162, 165, and 168 are SEQ ID NOs:6, 9, 12, 15, 18, 21,
24, 27, 30, 33, 36, 39, 42, 45, 48, 155, 158, 161, 164, 167, and
170, respectively. It should be noted that since nucleic acid
sequencing technology is not entirely error-free, SEQ ID NO:1 (as
well as other nucleic acid and protein sequences presented herein)
represents an apparent nucleic acid sequence of the nucleic acid
molecule encoding an ALN protein of the present invention.
[0045] Translation of SEQ ID NO:1, the coding strand of
nCfALN.sub.2057, as well as translation of SEQ ID NO:4, the coding
strand of nCfALN.sub.1152, which represents the coding region of
SEQ ID NO:1, each yields a protein of about 384 amino acids,
denoted herein as PCfALN.sub.384, the amino acid sequence of which
is presented in SEQ ID NO:2, assuming a first in-frame codon
extending from nucleotide 1 to nucleotide 3 of SEQ ID NO:4.
[0046] Translation of SEQ ID NO:7, the coding strand of
nCfCBP.sub.1128, as well as translation of SEQ ID NO:10, the coding
strand of nCfCBP.sub.1128, which represents the coding region of
SEQ ID NO:7, each yields a protein of about 272 amino acids,
denoted herein as PCfCBP.sub.272, the amino acid sequence of which
is presented in SEQ ID NO:8, assuming a first in-frame codon
extending from nucleotide 1 to nucleotide 3 of SEQ ID NO:10.
[0047] Translation of SEQ ID NO:13, the coding strand of
nCfNKAB.sub.1714, as well as translation of SEQ ID NO:16, the
coding strand of nCfNKAB.sub.978, which represents the coding
region of SEQ ID NO:13, each yields a protein of about 326 amino
acids, denoted herein as PCfNKAB.sub.326, the amino acid sequence
of which is presented in SEQ ID NO:14, assuming a first in-frame
codon extending from nucleotide 1 to nucleotide 3 of SEQ ID
NO:16.
[0048] Translation of SEQ ID NO:19, the coding strand of
nCfLGIC.sub.2240, as well as translation of SEQ ID NO:22, the
coding strand of nCfLGIC.sub.1707, which represents the coding
region of SEQ ID NO:19, each yields a protein of about 569 amino
acids, denoted herein as PCfLGIC.sub.569, the amino acid sequence
of which is presented in SEQ ID NO:20, assuming a first in-frame
codon extending from nucleotide 1 to nucleotide 3 of SEQ ID
NO:22.
[0049] Translation of SEQ ID NO:25, the coding strand of
nCfANON.sub.1429, as well as translation of SEQ ID NO:28, the
coding strand of nCfANON.sub.1194, which represents the coding
region of SEQ ID NO:25, each yields a protein of about 398 amino
acids, denoted herein as PCfANON.sub.398, the amino acid sequence
of which is presented in SEQ ID NO:26, assuming a first in-frame
codon extending from nucleotide 1 to nucleotide 3 of SEQ ID
NO:28.
[0050] Translation of SEQ ID NO:31, the coding strand of
nCfMALV.sub.765, as well as translation of SEQ ID NO:34, the coding
strand of nCfMALV.sub.762, which represents the coding region of
SEQ ID NO:31, each yields a protein of about 327 amino acids,
denoted herein as PCfMALV.sub.254, the amino acid sequence of which
is presented in SEQ ID NO:32, assuming a first in-frame codon
extending from nucleotide 1 to nucleotide 3 of SEQ ID NO:34.
[0051] Translation of SEQ ID NO:37, the coding strand of
nCfOSD.sub.604, as well as translation of SEQ ID NO:34, the coding
strand of nCfOSD.sub.405, which represents the coding region of SEQ
ID NO:37, each yields a protein of about 135 amino acids, denoted
herein as PCfOSD.sub.135, the amino acid sequence of which is
presented in SEQ ID NO:38, assuming a first in-frame codon
extending from nucleotide 1 to nucleotide 3 of SEQ ID NO:40.
[0052] Translation of SEQ ID NO:43, the coding strand
ofNMDA.sub.1227, as well as translation of SEQ ID NO:46, the coding
strand of nCfMDA.sub.738, which represents the coding region of SEQ
ID NO:43, each yields a protein of about 246 amino acids, denoted
herein as PCfNMDA.sub.246, the amino acid sequence of which is
presented in SEQ ID NO:44, assuming a first in-frame codon
extending from nucleotide 1 to nucleotide 3 of SEQ ID NO:46.
[0053] Translation of SEQ ID NO:153, the coding strand of
nCfCLBP1A.sub.633, as well as translation of SEQ ID NO:156, the
coding strand of nCfCLBP1A.sub.441, which represents the coding
region of SEQ ID NO:153, each yields a protein of about 147 amino
acids, denoted herein as PCfCLBP.sub.147, the amino acid sequence
of which is presented in SEQ ID NO:154, assuming a first in-frame
codon extending from nucleotide 1 to nucleotide 3 of SEQ ID
NO:156.
[0054] Translation of SEQ ID NO:162, the coding strand of
nCfCLBP2A.sub.631, as well as translation of SEQ ID NO:165, the
coding strand of nCfCLBP2A.sub.441, which represents the coding
region of SEQ ID NO:162, each yields a protein of about 147 amino
acids, denoted herein as PCfCLBP2A.sub.147, the amino acid sequence
of which is presented in SEQ ID NO:163, assuming a first in-frame
codon extending from nucleotide 1 to nucleotide 3 of SEQ ID
NO:165.
[0055] Translation of SEQ ID NO:1861, the coding strand of
nCfLGIC.sub.2739, as well as translation of SEQ ID NO:1864, the
coding strand of nCfLGIC.sub.2016, which represents the coding
region of SEQ ID NO:1861, each yields a protein of about 672 amino
acids, denoted herein as PCfLGIC.sub.672, the amino acid sequence
of which is presented in SEQ ID NO:1862, assuming a first in-frame
codon extending from nucleotide 1 to nucleotide 3 of SEQ ID
NO:1864.
[0056] Translation of SEQ ID NO:1867, the coding strand of
nCfNAH.sub.2080, as well as translation of SEQ ID NO:1870, the
coding strand of nCfNAH.sub.1824, which represents the coding
region of SEQ ID NO:1867, each yields a protein of about 608 amino
acids, denoted herein as PCfNAH.sub.608, the amino acid sequence of
which is presented in SEQ ID NO:1868, assuming a first in-frame
codon extending from nucleotide 1 to nucleotide 3 of SEQ ID
NO:1870.
[0057] Translation of SEQ ID NO:1872, the coding strand of
nCfCLIC.sub.2283, as well as translation of SEQ ID NO:1875, the
coding strand of nCfCLIC.sub.786, which represents the coding
region of SEQ ID NO:1872, each yields a protein of about 262 amino
acids, denoted herein as PCfCLIC.sub.262, the amino acid sequence
of which is presented in SEQ ID NO:1873, assuming a first in-frame
codon extending from nucleotide 1 to nucleotide 3 of SEQ ID
NO:1875.
[0058] Translation of SEQ ID NO:1882, the coding strand of
nCfPL2.sub.1477, as well as translation of SEQ ID NO:1885, the
coding strand of nCfPL2.sub.1359, which represents the coding
region of SEQ ID NO:1882, each yields a protein of about 453 amino
acids, denoted herein as PCfPL2.sub.453, the amino acid sequence of
which is presented in SEQ ID NO:1883, assuming a first in-frame
codon extending from nucleotide 1 to nucleotide 3 of SEQ ID
NO:1885.
[0059] Translation of SEQ ID NO:1887, the coding strand of
nCfPL3.sub.406, as well as translation of SEQ ID NO:1890, the
coding strand of nCfPL3.sub.243, which represents the coding region
of SEQ ID NO:1887, each yields a protein of about 81 amino acids,
denoted herein as PCfPL3.sub.81, the amino acid sequence of which
is presented in SEQ ID NO:1888, assuming a first in-frame codon
extending from nucleotide 1 to nucleotide 3 of SEQ ID NO:1890.
[0060] Translation of SEQ ID NO:1896, the coding strand of
nCfPL4.sub.1062, as well as translation of SEQ ID NO:1899, the
coding strand of nCfPL4.sub.285, which represents the coding region
of SEQ ID NO:1896, each yields a protein of about 285 amino acids,
denoted herein as PCfPL4.sub.285, the amino acid sequence of which
is presented in SEQ ID NO:1897, assuming a first in-frame codon
extending from nucleotide 1 to nucleotide 3 of SEQ ID NO:1899.
[0061] Translation of SEQ ID NO:1901, the coding strand of
nCfSVP.sub.1875, as well as translation of SEQ ID NO:1904, the
coding strand of nCfSVP.sub.530, which represents the coding region
of SEQ ID NO:1901, each yields a protein of about 530 amino acids,
denoted herein as PCfSVP.sub.530, the amino acid sequence of which
is presented in SEQ ID NO:1902, assuming a first in-frame codon
extending from nucleotide 1 to nucleotide 3 of SEQ ID NO:1904.
[0062] Translation of SEQ ID NO:1914, the coding strand of
nCfVGCC.sub.3126, as well as translation of SEQ ID NO:1917, the
coding strand of nCfVGCC.sub.2553, which represents the coding
region of SEQ ID NO:1914, each yields a protein of about 851 amino
acids, denoted herein as PCfVGCC.sub.851, the amino acid sequence
of which is presented in SEQ ID NO:1915, assuming a first in-frame
codon extending from nucleotide 1 to nucleotide 3 of SEQ ID
NO:1917.
[0063] Translation of SEQ ID NO:1919, the coding strand of
nCfAUP.sub.1181, as well as translation of SEQ ID NO:1922, the
coding strand of nCfAUP.sub.306, which represents the coding region
of SEQ ID NO:1919, each yields a protein of about 102 amino acids,
denoted herein as PCfAUP.sub.102, the amino acid sequence of which
is presented in SEQ ID NO:1920, assuming a first in-frame codon
extending from nucleotide 1 to nucleotide 3 of SEQ ID NO:1922.
[0064] Translation of SEQ ID NO:1924, the coding strand of
nCf7B2.sub.2161, as well as translation of SEQ ID NO:1927, the
coding strand of nCf7B2.sub.801, which represents the coding region
of SEQ ID NO:1924, each yields a protein of about 267 amino acids,
denoted herein as PCf7B2.sub.267, the amino acid sequence of which
is presented in SEQ ID NO:1925, assuming a first in-frame codon
extending from nucleotide 1 to nucleotide 3 of SEQ ID NO:1927.
[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.
1TABLE I SEQ ID NO: Name Genbank Homology Organism 63 2096-46
ATPase 6 D. melanogaster 64 2098-25 ATP synthase delta chain Sus
scrofa 65 2098-34 F1-ATPase epsilon-subunit Ipomoea batatas 66
2110-19 ATP synthase beta subunit Drosophila pseudoobscura 67
2113-15 ATP synthase delta chain, Sus scrofa 68 2180-31 ATP
synthase alpha subunit precursor Rattus rattus 69 2224-50
oligomysin sensitivity conferring protein D. melanogaster 70
2116-51 cysteine dioxygenase Homo sapiens 71 2116-55
pyrroline-5-carboxylate Homo sapiens dehydrogenase (P5CDh) 72
2124-17 AMP deaminase Homo sapiens 73 2138-38 ubiquitin Mus
musculus 74 2184-59 manganese superoxide dismutase Homo sapiens 75
2096-24 muscle LIM protein 1 D. melanogaster 76 2140-53 F25H5.1a C.
elegans 77 2176-41 Frazzled D. melanogaster 78 2223-11 LIMm
domain-containing protein C. elegans 79 2223-53 deleted in split
hand/split foot 1 (DSS1) Homo sapiens 80 2225-28 stranded-at-second
D. melanogaster 81 2099-61 histone H3 Spisula solidissima 82
2114-21 STE12 S. cereviciae 83 2117-4 Rad51 homolog Bombyx mori 84
2138-46 heat shock protein p27 D. immitis 85 2182-37 heat shock
protein 70 D. immitis 86 2211-32 BTB-II protein domain gene D.
melanogaster 87 2223-7 heat shock protein D. melanogaster 88
2224-17 heat shock protein 86 Homo sapiens 89 2225-16 POU domain
protein D. melanogaster 90 2225-18 nucleolin Xenopus laevis 91
2212-85 thyroid hormone receptor-associated Homo sapiens protein
complex component TRAP220 92 2211-21 T03D8.3 C. elegans 93 2223-67
hepatoma derived growth factor (HDGF) Mus musculus 94 2225-61
tyrosine hydroxylase type 1 (neuronal D. melanogaster form) 95
2097-7 sarco/endoplasmic reticulum-type D. melanogaster
Ca-2+-ATPase 96 2098-27 calcium-transporting ATPase D. melanogaster
97 2099-19 calcium channel alpha-1 subunit Aplysia californica 98
2120-5 P-type voltage-gated calcium channel Homo sapiens alpha 1
subunit homolog 99 2124-2 sarco/endoplasmic reticulum Procambarus
clarkii Ca2+-ATPase (SERCA) 100 2182-43 sulfonylurea receptor 2b
Mus musculus 101 2223-18 Sodium-Potassium-Chloride cotransporter D.
melanogaster 102 2223-63 sarco/endoplasmic reticulum-type D.
melanogaster Ca2(+)-ATPase 103 2224-13 similar to ABC transporters
C. elegans 104 2098-3 Camguk D. melanogaster 105 2101-9 UNC-89 C.
elegans 106 2132-31 arginine kinase Homarus gammarus 107 2141-51
casein kinase-II beta Oryctolagus cuniculus 108 2178-18
diacylglycerol kinase eta Cricetinae 109 2180-32 retinoid- and
fatty acid-binding D. melanogaser glycoprotein 110 2137-23
vitellogenin Aedes aegypti 111 2144-14 nuclear localization signal
spot 1 Mus musculus 112 2212-13 putative n- terminal
acetyltransferase S. cereviciae 113 2212-27 clathrin associated
protein AP47 Drosophila grimshawi 114 2223-28 O1
chloroquine-resistance protein Plasmodium falciparans 115 2224-14
vitellogenin Athalia rosae 116 2224-15 antigen NY-CO-3 Homo sapiens
117 2225-24 carbonic anhydrase C. elegans 118 2225-58 yk500f6.3 C.
elegans 119 2225-76 unknown Homo sapiens 120 2224-86 BmP109
(cerebroside sulfate activator Bombyx mori protein family) 121
2225-23 intersectin Homo sapiens 122 2170-16 chemical-sense-related
Phormia regina lipophilic-ligand-binding protein 123 2176-2
olfactory receptor protein 2.4 Danio rerio 124 2212-63 olfactory
receptor Xenopus laevis 125 2224-77 inner mitochondrial membrane
Homo sapiens translocase Tim23 126 2225-12 sodium-dependent
multi-vitamin Rattus norvegicus transporter 127 2225-42 ribophorin
I Rattus norvegicus 128 2101-59 phosphate carrier protein C.
elegans 129 2132-38 proteinase inhibitor Locusta migratoria 130
2174-72 HE4 protein Homo sapiens 131 2211-48 spermatogenic
cell/sperm-associated Rattus norvegicus Tat-binding homologue 132
2110-23 Gcap1 gene product Mus musculus 133 2116-64 toll protein D.
melanogaster 134 2124-3 tuberin (TSC2) gene Homo sapiens 135
2178-55 RAS-like protein Gallus gallus 136 2223-35 Rho1 gene
product D. melanogaster 137 2224-82 paxillin Homo sapiens 138
2225-44 adenylyl cyclase-associated protein (CAP) Homo sapiens 139
2225-80 adenylate kinase Gallus gallus 140 2110-52
hydroxyproline-rich glycoprotein Phaseolus vulgaris 141 2115-49
mitogen inducible gene mig-2 Homo sapiens 142 2116-5 F52H3.5 C.
elegans 143 2172-89 12D3 antigen Babesia bovis 144 2178-20
frameshift P. falciparum 145 2178-81 KIAA0066 Homo sapiens 146
2182-16 Y57G11C.4 C. elegans 147 2182-53 C16C10.5 C. elegans 148
2211-8 Unknown Homo sapiens 149 2211-31 hopothetical protein
Arabidopsis thaliana 150 2223-54 ORF YNL207w S. cereviciae 151
2224-94 14.3 kDa perchloric acid soluble protein Capra hircus 152
2225-36 EST clone C. elegans 1719 2228-2 BIGH3 H. sapiens 1720
2228-5 H protein H. sapiens 1721 2228-8 ubiquinol-cytochrome c
reductase Schizosaccharomyces pombe 1722 2228-11 similar to
mitochondrial ATPase inhibitors C. elegans 1723 2228-16 Putative
enzyme E. coli 1724 2228-18 Ribosomal protein L7A Drosophila 1725
2228-22 Troponin-I wings up A Drosophila 1726 2228-25 tls gene
product E. coli 1727 2228-27 YCR521 gene product Saccharomyces
cerevisiae 1728 2228-28 putative transport system permease E. coli
protein 1729 2228-32 SapA protein E. coli 1730 2228-34 Putative
protein Arabidopsis thaliana 1731 2228-37 Ada E. coli 1732 2228-39
Titin H. sapiens 1733 2228-42 adenylosuccinate synthetase Mus
musculus 1734 2228-43 transfer RNA-Ala synthetase B. mori 1735
2228-44 C4 zinc finger DNA-binding protein Drosophila 1736 2228-48
heme A: farnesyltransferase H. sapiens 1737 2228-51 URF 4L (aa
1-96) Drosophila 1738 2228-53 DOLICHOL-PHOSPHATE E. coli
MANNOSYLTRANSFERASE 1739 2228-58 troponin-T Drosophila 1740 2228-59
protein disulfide isomerase Drosophila 1741 2228-63 orf,
hypothetical protein E. coli 1742 2228-66 ilvl polypeptide E. coli
1743 2228-68 orf, hypothetical protein E. coli 1744 2228-72
Respiratory nitrate reductase 1 alpha E. coli chain 1745 2228-77
homolog of virulence factor E. coli 1746 2228-84 ORF o164 E. coli
1747 2228-91 nuclear protein E3-3 orf1 Rattus norvegicus 1748
2245-66 Troponin C Drosophila 1749 2245-70 Predicted secreted
protein Plasmodium falciparum 1750 2245-72 Cytochrome C-1 H.
sapiens 1751 2245-75 rpoB Plasmodium falciparum 1752 2245-78
sarco(endo) plasmic reticulum-type Helliothis virescens calcium
ATPase 1753 2246-31 Ras-related GTP-binding protein H. sapiens 1754
2246-57 Similar to inositol 1,4,5-triphosphate C. elegans receptor
1755 2246-61 reverse transcriptase-like protein Aedes aegypti 1756
2247-13 polyprotein Drosophila 1757 2247-14 ORF2 for putative
reverse transcriptase Drosophila 1758 2247-42 Asparaginyl tRNA
Synthetase H. sapiens 1759 2247-44 calcium binding protein
Drosophila 1760 2247-58 similar to Fibronectin type III domain C.
elegans 1761 2247-62 reverse transcriptase Drosophila 1762 2247-65
gag-like protein Culex pipiens 1763 2247-79 L-3-phosphoserine
phosphatase H. sapiens 1764 2247-80 esterase E4 Myzus persicae 1765
2247-89 Similar to aldehyde dehydrogenase C. elegans 1766 2248-76
O-44 protein Rattus sp. 1767 2248-85 cDNA isolated for this protein
using a H. sapiens monoclonal antibody directed against the p27k
prosomal protein 1768 2249-3 Projectin Drosophila 1769 2249-5
ORF_ID:o312#14 E. coli 1770 2249-9 Heat shock protein 60 Culicoides
variipennis 1771 2249-11 enigma protein H. sapiens 1772 2249-12
alpha, alpha-trehalose glucohydrolase Oryctolagus cuniculus 1773
2249-13 small GTP binding protein Drosophila 1774 2249-14
Spermidine/putrescine transport system E. coli permease 1775
2249-19 nueroendocrine-specific protein C H. sapiens 1776 2249-21
a-agglutinin core subunit Saccharomyces cerevisiae 1777 2249-24
KIAA0337 H. sapiens 1778 2249-34 su(wa) protein Drosophila 1779
2249-42 regulator of kdp operon E. coli 1780 2249-59 No definition
line found C. elegans 1781 2249-60 proline oxidase Drosophila 1782
2249-62 Formate acetyltransferase E. coli 1783 2249-70 similar to
HECT-domain C. elegans 1784 2249-75 PHOSPHORIBOSYLFORMYLGLYCINAM E.
coli IDINE CYCLO-LIGASE 1785 2249-77 Hypothetical 38.5 kd protein
in agal-mtr E. coli intergenic region precursor 1786 2249-85 D4L
Variola virus 1787 2249-87 similar to isocitrate dehydrogenase C.
elegans 1788 2250-6 Fii (head-tail joining; 117) Bacteriophage
Lambda 1789 2250-7 possible NAGC-like transcriptional E. coli
regulator 1790 2250-10 cysteine string protein Bos taurus 1791
2250-13 Tol B protein E. coli 1792 2250-14 6-phosphogluconate
dehydratase E. coli 1793 2250-15 6-phosphogluconate dehydratase E.
coli 1794 2250-22 PSST subunit of the NADH: ubiquinone Bos taurus
oxidoreductase 1795 2250-30 sol i 3 antigen Solenopsis invicta 1796
2250-36 predicted using Genefinder; similar to C. elegans tRNA
synthetases class I (E and Q 1797 2250-37 PNP H. sapiens 1798
2250-42 ORF_ID:o331#2 E. coli 1799 2250-44 Extensin E. coli 1800
2250-47 ORF o654 E. coli 1801 2250-48 Gcap1 gene product Mus
musculus 1802 2250-52 similar to human MLH1 on chromosome Mus
musculus 3p21 1803 2250-53 Hypothetical 27.6 kd protein in hpt-panD
E. coli intergenic region. 1804 2250-58 UmuC protein E. coli 1805
2250-61 dJ134E15.1 (Blimp-1 H. sapiens 1806 2250-63 ribosomal
protein L23-related product Rattus rattus homolog 1807 2250-65
hypothetical protein MJ1143 E. coli 1808 2250-68 HI0025 homolog E.
coli 1809 2250-77 R34094_1 H. sapiens 1810 2250-78 erythrocyte
binding protein Plasmodium yoelii 1811 2250-79 fosmidomycin
resistance protein E. coli 1812 2250-81 cyclophilin 1 Drosophila
1813 2250-83 putative glutamine synthetase E. coli 1814 2251-3 J
(tail:host specificity; 1132) Bacteriophage Lambda 1815 2251-5
Molybdopterin biosynthesis MoeB protein E. coli 1816 2251-6 Fo-ATP
synthase subunit b Drosophila 1817 2251-9 citrate lyase alpha chain
E. coli 1818 2251-10 cuticle protein ACP65A Drosophila 1819 2251-13
H repeat-associated protein in rhsC E. coli 3'region (orf-h3 1820
2251-20 glycine-rich protein Arabadopsis thaliana 1821 2251-23
2-oxoglutarate dehydrogenase precursor H. sapiens 1822 2251-29 NFX1
H. sapiens 1823 2251-32 ebgR product, repressor E. coli 1824
2251-41 neural protein Drosophila 1825 2251-45 similar to
unidentified ORF E. coli 1826 2251-46 NADH:ubiquinone
oxidoreductase b17.2 Bos taurus subunit 1827 2251-49 tyrosine
kinase Drosophila 1828 2251-50 coded for by C. elegans cDNA
yk89e9.5 C. elegans 1829 2251-57 H (tail component; 853)
Bacteriophage Lambda 1830 2251-60 Lysyl tRNA Synthetase Drosophila
1831 2251-62 7,8-diamino-pelargonic acid E. coli aminotransferase
1832 2251-64 actin related protein Drosophila 1833 2252-6
discs-large tumor suppressor Drosophila 1834 2252-16
S-adenosylmethionine decarboxylase E. coli 1835 2252-17 F52H3.5 E.
coli 1836 2252-21 translationally controlled tumor protein
Oryctolagus cuniculus 1837 2252-31 GTP binding protein Rattus
rattus 1838 2252-34 mitochondrial porin transcript 1 Drosophila
1839 2252-38 cuticle protein Manduca sexta 1840 2252-39 Similarity
to Rat CD63 antigen C. elegans 1841 2252-41 similar to S.
cerevisiae Lpg20p E. coli 1842 2252-48 cut E E. coli 1843 2252-61
Histone H3 Spisula solidissima 1844 2252-66 ea10 (ssb; 122)
Bacteriophage Lambda 1845 2252-71 Mao C protein E. coli 1846
2252-72 miniparomyosin Drosophila 1847 2252-73 pherophorin-S Volvox
carteri 1848 2252-80 cyclophilin Mus musculus 1849 2252-84
alternate gene name yhhG E. coli 1850 2222-20 nucleoporin Nup98 rat
1851 2222-21 hypothetical protein Escherichia coli 1852 2222-36
ribosomal protein S11 human 1853 2222-39 hypothetical protein
PFB0315w Plasmodium falciparans 1854 2222-50
serine/threonine-specific protein k. Plasmodium falciparans 1855
2222-58 hypothetical protein C25E10.9 C. elegans 1856 2222-64
transporting ATP synthase bovine 1857 2222-94 tricarboxylate
carrier rat 1858 2218-95 anoxia upregulated protein Drosophila
melanogaster
[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.
2TABLE II SEQ ID NO: Name GenBank Homology Organism 171 2094-23
mitochondrian ATP synthase, alpha subunit Drosophila melanogaster
172 2104-20 mitochondrial ATP synthase Drosophila melanogaster 173
2105-14 ATP synthase gamma-subunit Homo sapiens 174 2167-72
oligomysin sensitivity conferring protein Drosophila melanogaster
175 2179-20 ATPase 6 Drosophila melanogaster 176 2193-60 ATP
synthase subunit B Schizaphis graminum 177 2229-41 ATP synthase
alpha subunit D. melanogaster 178 2231-35 9 kD basic protein D.
melanogaster 179 2231-47 ATP synthase alpha-subunit Bos taurus 180
2232-95 mitochondrial ATP synthase subunit 9 Homo sapiens 181
2084-56 Late embryogenesis abundant protein Picea glauca 182
2084-36 TGF-beta masking protein/stranded at second Drosophila
melanogaster 183 2086-2 Argonaute protein Arabidopsis thaliana 184
2196-92 like Drosophila HMPB homeotic C. elegans proboscipedia
protein 185 2092-27 DMDHEM2 Drosophila melanogaster 186 2094-21
SeID protein Drosophila melanogaster 187 2106-11 Unr Rattus
norvegicus 188 2231-15 cno (canoe) D. melanogaster 189 2230-79 ALR
homologue D. melanogaster 190 2232-42 saxophone serine-threonine
kinase receptor D. melanogaster 191 2232-68 selenophosphate
synthetase D. melanogaster 192 2088-11 MMTAX107, TAX responsive
element Mus musculus binding protein 193 2089-2 cs Dna J-1 Cucumis
sativus 194 2090-7 Lethal (2) TID Drosophila melanogaster 195
2102-33 monocytic leukaemia zinc finger protein Homo sapiens 196
2105-26 orf1 5'of EpoR Mus musculus 197 2106-6 contains similarity
to EGF-1 C. elegans 198 2106-9 HSP70 protein Ceratitis capitata 199
2084-60 82 kD heat shock protein Drosophila pseudobscura 200
2108-59 PAR domain protein Drosophila melanogaster 201 2156-34
yk29g12.3 C. elegans 202 2161-17 segmentation protein Drosophila
melanogaster 203 2162-28 heat shock protein 70, hsp70A2 Anopheles
albimanus 204 2187-18 Heat shock protein 70 Anopheles albimanus 205
2173-77 Heat shock protein hsp70 D. melanogaster 206 2165-30
nucleolar protein Drosophila melanogaster 207 2165-59 contains
similarity to C4-type zinc fingers C. elegans 208 2177-80 zinc
finger protein Mus musculus 209 2181-45 PAR domain protein 1
Drosophila melanogster 210 2185-9 Heat shock protein-70 Anopheles
albimanus 211 2185-82 segmentation protein Drosophila melanogaster
212 2188-33 transcriptional repressor protein Drosophila
melanogaster 213 2203-18 Mastermind Drosophila virilis 214 2205-82
high mobility group protein 1a Chironomus tentans 215 2230-26 DNA
repair protein D. melanogaster 216 2230-71 homologue of seven in
absentia Homo sapiens 217 2230-89 nuclear speckle-type protein,
SPOP Homo sapiens 218 2230-96 heat shock protein D. melanogaster
219 2231-7 hypothetical protein S. pombe 220 2231-38 Rad51 homolog
Bombyx mori 221 2231-81 DNA repair protein D. melanogaster 222
2232-2 cellular nucleic acid binding protein Xenopus laevis 223
2234-63 heat shock protein 70 Trichoplusia ni 224 2232-77
actin-binding double-zinc-finger protein Homo sapiens (abLIM) 225
2234-78 DNA-binding protein isoform I D. melanogaster 226 2084-48
Allantoinase Rana catesbeiana 227 2085-22 beta-glucuronidase E.
coli 228 2094-24 prolidase = peptidaseD/imidopeptidase Mus musculus
229 2088-43 branched chain alpha-keto acid Bos taurus dehydrogenase
E1-beta subunit 230 2086-29 3-hydroxyisobutyrate dehydrogenase
Dictyostelium discoideum 231 2088-5 Rab 5c protein Canis familiaris
232 2095-17 cytochrome P-450 Heliothis virescens 233 2102-16
carbamoyl phosphate synthetase II Plasmodium falciparans 234
2102-48 NADPH cytochrome P450 reductase Musca domestica 235 2104-15
branched chain alpha-keto acid Rattus norvegicus dehydrogenase 236
2106-5 Metallothionein Strongylocentrotus purpuratus 237 2106-47
peroxidoxin-1 Dirofilaria immitis 238 2107-17 tetracycline
transporter-like protein Mus musculus 239 2107-58 allergen Bla g 5
(glutathione-S-transferase) Blattella germanica 240 2156-58 HAL-3
homologue Arabidopsis thaliana 241 2195-90 aminoacyclase-1 Homo
sapiens 242 2171-55 NADPH-ferrihemoprotein reductase Drosophila
melanogaster 243 2169-30 hypothetical protein Synechocystis sp 244
2169-52 insulin degrading enzyme Drosophila melanogaster 245
2177-64 3-hydroxyisobutyrate dehydrogenase Rattus norvegicus 246
2181-69 Endonexin Bos taurus 247 2138-25 glutamate dehydrogenase
Drosophila melanogaster 248 2230-28 glutathione -S-transferase
Anopheles gambiae 249 2191-8 lactase-phlorizin hydrolase Rattus
rattus 250 2193-52 cytochrome P450 Heloithis virescens 251 2202-35
glutathione-S-transferase Anopheles gambiae 252 2229-77
glutathione-S-transferase Anopheles gambiae 253 2229-81 urate
oxidase D. melanogaster 254 2231-42 superoxide dismutase Cervus
elaphus 255 2232-74 allergen Bla g 5 Blattella germanica 256
2234-42 glutathione reductase family Musca domestica 257 2087-8
cystic fibrosis transmembrane Homo sapiens conductance regulator
258 2087-23 Nervous system antigen 2 Drosophila melanogaster 259
2091-56 adenosine triphosphatase Homo sapiens 260 2094-20 sodium
pump, alpha suhbunit Ctenocephalides felis 261 2095-51 similar to
Hrs C. elegans 262 2103-24 N-methyl-D-aspartate receptor-associated
Drosophila melanogaster protein 263 2105-55 inward rectifying K
channel Sus scrofa 264 2105-63 EF-hand Ca2+ binding protein p22
Rattus norvegicua 265 2106-62 Dents disease candidate gene product
Homo sapiens 266 2167-50 PKD1 (polycystic kidney disease 1) Fugu
rubripes 267 2185-37 copper-transporting ATPase Archaeoglobus
fulgidus 268 2193-29 TrkG Potassium transport protein E. coli 269
2195-33 silicon transporter Cylindrotheca fusiformis 270 2202-16
similarity to human sulfate anion transporter C. elegans 271 2230-2
sulfate transporter Arabidopsis thaliana 272 2230-69 mitochondrial
porin D. melanogaster 273 2231-22 muscarinic acetylcholine receptor
D. melanogaster 274 2231-24 p97 subunit of 15S Mg(2+)- ATPase
Xenopus laevis 275 2231-32 anion transporting ATPase Aquifex
aeolicus 276 2231-70 sulfate permease Schizosaccharomyces pombe 277
2231-94 putative Na/H exchanger S.pombe 278 2233-6 plasma membrane
Ca2+-ATPase 2 Mus musculus 279 2233-24 chloride channel gene, CLIC2
Homo sapiens 280 2085-61 beta-type protein kinase C Bos taurus 281
2089-20 cGMP-dependent protein kinase Drosophila melanogaster 282
2092-12 Btk Homo sapiens 283 2093-64 Receptor-like protein tyrosine
phosphatase Drosophila melanogaster 284 2095-31 frt (fms-related
tyrosine kinase gene) Homo sapiens 285 2094-58 casein kinase II
beta Oryctolagus cuniculus 286 2103-54 ORF YGL084c Saccharomyces
cerevisiae 287 2106-42 protein phosphatase epsilon subunit Homo
sapiens 288 2156-5 serine/threonine kinase Rattus norvegicus 289
2157-95 cGMP-dependent protein kinase Drosophila melanogaster 290
2165-80 ABL gene product Gallus gallus 291 2165-63 diadenosine
tetraphosphatase Homo sapiens 292 2167-17 adenylate cyclase S.
cereviciae 293 2177-44 serine/threonine kinase C. elegans 294
2188-16 weakly similar to serine/threonine kinase C. elegans 295
2191-60 carbohydrate kinase, pfkB family Archaeoglobus fulgidus 296
2195-22 protein kinase Drosophila melanogaster 297 2196-30
calcium-dependent protein kinase A. thaliana 298 2205-83 protein
kinase/endoribonulcease (IRE1) Homo sapiens 299 2205-87 receptor
tyrosine phosphatase Hirudo medicinalis 300 2229-11
magnesium-dependent calcium Bos taurus inhibitable phosphatase 301
2229-29 phosphoglycerate kinase Schistosoma mansoni 302 2229-74
pyruvate kinase D. melanogaster 303 2230-55 serine/threonine
specific protein phosphatase 4 D. melanogaster 304 2230-57 stress
activated MAP kinase kinase 3 D. melanogaster 305 2231-64 alkaline
phosphatase D. melanogaster 306 2231-91 olynucleotide phosphorylase
Yersinia enterocolitica 307 2232-43 protein kinase PkwA
Thermomonospora curvata 308 2234-94 serine/threonine kinase ULK1
Homo sapiens 309 2085-18 Pyridoxamine phosphate oxidase C. elegans
310 2094-13 sphingomyelin phosphodiesterase Mus musculus 311
2105-47 apolipoprotein E receptor 2 Homo sapiens 312 2092-38
squalene synthetase Homo sapiens 313 2094-25 fatty acid synthetase
Rattus norvegicus 314 2089-32 coproporphyrinogen oxidase Homo
sapiens 315 2085-46 HADHB mitochondrial trifunctional protein Homo
sapiens beta subunit 316 2104-56 pyridoxal kinase Homo sapiens 317
2107-30 Phosphomevalonate kinase Homo sapiens 318 2154-70 very-long
chain acyl-CoA dehydrogenase Mus musculus 319 2191-85 stearyl-CoA
desaturase Cyprihnus carpio 320 2192-44 Very-long-chain Acyl-CoA
dehydrogenase Rattus norvegicus 321 2195-55 Similar to LDL
receptor-related protein C. elegans 322 2229-82 lipase-3 D.
melanogaster 323 2231-59 Phosphatidylethanolamine-binding protein
Macaca fascicularis 324 2233-25 similarity to C. elegans yeast
ethanolaminephosphotransf- erase 325 2233-41 cellular retinoic acid
binding protein Manduca sexta (mCRABP) 326 2087-61 I allergen
Lepidoglyphus destructor 327 2087-41 chloroquine resistance
candidate protein Plasmodium falciparum 328 2089-51 Xenopus Bf B
Xenopus laevis 329 2086-58 repeat organellar protein Plasmodium
falciparum 330 2090-45 heat shock cognate protein Drosophila
melanogaster 331 2104-23 40 kDa heat shock chaperone protein
Deinococcus 332 2107-26 Luciferase Photuris pennsylvanica 333
2162-46 F20D1.9 C. elegans 334 2162-49 PKR inhibitor P58 Bos taurus
335 2162-93 GroES homologue Ricketsia 336 2171-46 NH2 terminus
uncertain Leishmania tarentolae 337 2089-10 beta adaptin Drosophila
melanogaster 338 2229-24 non-functional folate binding protein Homo
sapiens 339 2229-25 calmodulin B Halocynthia roretzi 340 2229-31
putative T1/ST2receptor binding protein C. elegans 341 2229-36
alpha-crystallin cognate protein 25 Plodia interpunctella 342
2229-40 Defensin Apis mellifera 343 2229-86 glutamate-ammonia
ligase D. melanogaster 344 2231-49 melanoma-associated antigen
ME491 Homo sapiens 345 2231-76 histone C Drosophila virilis 346
2232-65 translationally controlled tumor protein Oryctolagus
cuniculus 347 2232-84 Apyrase Aedes aegypti 348 2232-85 KIAA0124
Homo sapiens 349 2233-59 Glutamine-dependent C. elegans
carbamoyl-phosphate synthase 350 2233-86 ANG12 precursor Anopheles
gambiae 351 2234-11 tissue specific secretory protein Pan
troglodytes 352 2234-76 methionine adenosyltransferase D.
melanogaster 353 2089-13 Synaptic vessicle protein 2 form B Rattus
norvegicus 354 2159-52 glycoprotein 56 Rattus norvegicus 355 2084-6
CLN3; homologue of the gene underlying Mus musculus Batten disease
356 2085-10 Amphiphysin Gallus gallus 357 2156-39 glycoprotein 55
Rattus norvegicus 358 2104-59 Transmembrane transporter Discopyge
ommata 359 2105-9 insect intestinal mucin II Trichoplusia ni 360
2106-14 kinesin-like protein D. melanogaster 361 2107-45 Lazarillo
precursor Schistocerca americana 362 2156-3 clathrin-associated
protein Mus musculus 363 2161-46 neural variant mena + protein Mus
musculus 364 2171-92 Malvolio Drosophila melanogaster 365 2175-18
homolog of SYT - synaptotagmin Mus musculus 366 2177-10 GABA
receptor subunit (Rdl) Aedes aegypti 367 2181-10 neurexin IV
Drosophila melanogaster 368 2191-92 synaptic vessicle protein 2B
Rattus norvegicus 369 2229-18 Synaptic vessicle protein 2A Rattus
norvegicus 370 2194-38 gamma-subunit of mouse nerve growth factor
Mus musculus 371 2230-60 lin-7-C Rattus norvegicus 372 2230-81 PDZ
domain protein Homo sapiens 373 2234-5 Gcap1 gene product Mus
musculus 374 2234-55 Gcap1 gene product Mus musculus 375 2234-71
Gcap1 gene product Mus musculus 376 2085-34 Liver-specific
transport protein Rattus norvegicus 377 2087-15 polyspecific
organic cation transporter Homo sapiens 378 2204-80 transmembrane
transporter Discopyge ommatta 379 2093-39 liver-specific transport
protein Rattus norvegicus 380 2093-46 similar to monocarboxylate
transporter family C. elegans 381 2092-22 similar to matrin F/G C.
elegans 382 2103-50 Unknown Drosophila melanogaster 383 2103-51
organic cation transporter Ratttus norvegicus 384 2197-35 renal
organic cation transporter Oryctolagus cuniculus 385 2156-17
sulfate anion transporter Manduca sexta 386 2166-84 LX1 Mus
musculus 387 2167-94 MCT (monocarboxylate transporter) Homo sapiens
388 2196-83 renal organic cation transporter Oryctolagus cuniculus
389 2229-83 similarity to monocarboxylate transporter 1 C. elegans
390 2231-89 Golgi 4-transmembrane spanning transporter Mus musculus
MTP 391 2158-8 phosphate carrier protein C. elegans 392 2085-14
ADP/ATP translocase Drosophila melanogaster 393 2085-17
Na+-dependent inorganic Drosophila melanogaster phosphatase
cotransporter 394 2088-38 ADP/ATP translocase Bos taurus 395
2092-50 ADP/ATP translocase Drosophila melanogaster 396 2104-21
Na(+)-dependent inorganic Drosophila melanogaster phosphate
cotransporter 397 2121-55 phosphate carrier protein C. elegans 398
2105-64 phosphate carrier protein Homo sapiens 399 2102-6 ZK512.6
C. elegans 400 2108-27 mitochondrial phosphate carrier protein Homo
sapiens 401 2194-63 mitochondrial phosphate transporter Rattus
norvegicus 402 2196-14 phosphate/triose-phospha- te C. elegans
translocator precursor 403 2204-11 EST clone D. melanogaster 404
2085-16 Chymotrypsin I Anopheles gambiae 405 2085-54 Chymotrypsin
II Anopheles gambiae 406 2086-12 Plasminogen Homo sapiens 407
2086-18 Trypsin eta Drosophila melanogaster 408 2090-21 Trypsin
Manduca sexta 409 2092-15 Alp1 Cochilobolus carbonum 410 2102-11
vitellin-degrading protease Bombyx mori 411 2102-17 Chymotrypsin II
Anopheles gambiae 412 2102-51 chymotrypsin-like protease Anopheles
gambiae 413 2103-31 Beta trypsin Drosophila erecta 414 2107-22
Factor IX Rattus norvegicus 415 2108-29 Trypsin Anopheles stephensi
416 2157-15 Trypsin Choristoneura fumiferana 417 2160-34
Aminopeptidase Synechocystis 418 2160-36 E01G6.1 C. elegans 419
2103-62 plasminogen activator inhibitor 2 Mus musculus 420 2167-36
factor IX Oryctolagus cuniculus 421 2167-67 Alp1 Cochliobolus
carbonum 422 2169-51 Trypsin Aedes aegypti 423 2181-27 Chymotrypsin
BII Penaeus vannamei 424 2185-69 plasma prekallikrein Homo sapeins
425 2187-20 pre-procathepsin L Paragonimus westermani 426 2188-45
vitellin-degrading protease Bombyx mori 427 2192-91 late trypsin
precourser Culex pipiens quinquefasciatus 428 2196-10 SPC2
Branchiostoma californiensis 429 2196-88 Trypsin Anopheles
stephensi 430 2204-9 carnitine/choline acetyltransferase C. elegans
431 2229-7 iota trypsin D. melanogaster 432 2229-22 Trypsin
Anopheles gambiae 433 2229-89 Trypsin Anopheles gambiae 434 2229-94
late trypsin precourser Culex pipiens quinquefasciatus 435 2230-59
Chymotrypsin 1 Anopheles gambiae 436 2230-67 carboxypeptidase A
Drosophila heteroneura 437 2231-62 aminopeptidase N Sus scxrofa 438
2231-74 limulus factor C serine protease Tachypleus tridentatus 439
2232-15 cysteine proteinase Sitophilus zeamais 440 2232-25
Carboxypeptidase Simulium vitatum 441 2232-33 putative aspartic
protease Brassica oleracea 442 2233-46 aminopeptidase N
Pleuronectes americanus 443 2233-85 chymotrypsin 1 Anopheles
gambiae 444 2233-90 Trypsin Anopheles stephensi 445 2233-94
preprechymotrypsin 1 Penaeus vannamei 446 2234-29 chymotrypsin-like
protease precursor Aedes aegypti 447 2234-58 Putative C. elegans
448 2234-61 carboxylesterase precursor Aphis gossypii 449 2234-68
serine protease inhibitor I Schistocerca gregaria 450 2084-35
Integral membrane protein Mus musculus 451 2086-45 similar to
beta-ureidopropionase of Rat C. elegans 452 2087-54 Cyclin Mus
musculus 453 2088-22 Esp 8
Mus musculus 454 2091-16 contains similarity to EGF-like domains C.
elegans 455 2091-29 multiple exostosis-like protein Homo sapiens
456 2091-30 apoptosis 1 inhibitor Drosophila melanogaster 457
2092-33 KIAA0023 (putitive oncogene) Homo sapiens 458 2095-35 G
coupled receptor C. elegans 459 2095-3 Go (heterotrimeric guanyl
nucleotide Manduca sexta binding protein alpha subunit) 460 2085-4
gp 150 protein Drosophila melanogaster 461 2103-28 leukotriene A4
hydrolase Rattus sp. 462 2105-62 putitive orf Homo sapiens 463
2107-6 activator protein Drosophila melanogaster 464 2107-28
platelet-endothelial tetraspan antigen 3 Homo sapiens 465 2189-3
oligopeptidase A (prlC) Haemopholis influenzea 466 2156-54
fibroblast growth factor receptor Xenopus laevis 467 2160-92
contains similarity to EGF-like domains C. elegans 468 2160-65 weak
similarity to the drosophila hyperplastic C. elegans disc protein
469 2165-53 inositol triphosphate receptor Rattus norvegicus 470
2166-22 placental protein 11 Homo sapiens 471 2166-92 elongation
factor 1 alpha-like Drosophila melanogaster 472 2181-34 DSch
Drosophila melanogaster 473 2192-65 STAM, signal transducing
adaptor molecule Homo sapiens 474 2194-24 ATPases associated with
various cellular Arabidopsis thaliana activities (AAA family) 475
2196-75 similar to cell division control protein C. elegans 476
2230-38 EST clone S. cereviciae 477 2230-39 NTPase D. melanogaster
478 2230-66 adenylyl cyclase aggregation protein Dictyostelium
discoideum 479 2230-80 sphingomyelin phosphodiesterase C. elegans
480 2231-29 nuclear antigen H731 Homo sapiens 481 2231-40
suppressor of actin mutation 2 Homo sapiens 482 2231-66 DET1
Arabidopsis thaliana 483 2232-7 Calreticulin D. melanogaster 484
2232-38 activator protein D. melanogaster 485 2232-69 ornithine
decarboxylase Gallus gallus 486 2233-32 similar bHLH-PAS D.
melanogaster 487 2233-45 rab1 D. melanogaster 488 2234-2 C10A gene
product Mus musculus 489 2234-72 QM homolog D. melanogaster 490
2084-17 Integral membrane protein Herpesvirus-2 491 2091-4
endomembrane protien EMP70 precourser Arabidopsis thaliana isolog
492 2102-45 Ylr251wp Saccharomyces cerevisiae 493 2162-68 220 kDa
silk protein Chironomus thummi 494 2160-47 precursor HT7 protein
Gallus gallus 495 2161-12 peritrophin 95 precourser Lucilia cuprina
496 2161-15 yk86g11.5 C. elegans 497 2171-12 51A surface protein
Paramecium tetraurelia 498 2173-18 hypothetical - mitochondrial
membrane Schizosaccharomyces transport protein pombe 499 2087-32
est sequence C. elegans 500 2091-19 Similar to P. aeruginosa
hypothetical protein C. elegans 501 2192-86 tyrosine kinase
Drosophila melangaster 502 2086-42 M04B2.4 C. elegans 503 2088-16
glycoprotein 330 C. elegans/Human 504 2088-39 EST sequence
Arabidopsis thaliana 505 2088-57 Yer 126cp Saccharomyces cereviciae
506 2089-25 similar to S. cereviciae hypothetical C. elegans
protein YKL166 507 2090-3 EST sequence Saccharomyces cereviciae 508
2090-53 EST sequence C. elegans 509 2095-20 Chloroplast ORF
Marchantia polymorpha 510 2102-28 similar to S. cerevisiae
hypothetical C. elegans protein YKL166 511 2102-55 D1054.3 C.
elegans 512 2102-58 ZC513.5 gene product C. elegans 513 2105-44 E
1087 protein Saccharomyces cerevisiae 514 2109-24 F11C1.5 C.
elegans 515 2154-21 disulfide-like protein Acanthamoeba castellanii
516 2156-6 ZK470.1 C. elegans 517 2156-18 BIIIA3 Ovis aries 518
2156-27 AFR1 S. cereviciae 519 2165-94 COS41.8 Ciona intestinalis
520 2167-65 EST sequence, function unknown C. elegans 521 2171-93
KIAA0160 Homo sapiens 522 2175-45 ORF YJR83.18 S. cereviciae 523
2185-66 rps4 Plasmodium falciparum 524 2195-40 C27C12.4 C. elegans
525 2196-20 glycoprotein A Pneumocystis carinii 526 2205-89 BKRF1
encodes EBNA-1 protein Epstein Barr virus 527 2229-19 D4L Variola
virus 528 2230-35 KIAA0747 Homo sapiens 529 2231-8 I3 Mus musculus
530 2231-78 unknown protein Arabidopsis thaliana 531 2232-49
Similarity to Yeast hypothetical 52.9 KD C. elegans protein 532
2232-52 tetratricopeptide repeat protein (tpr2) Homo sapiens 533
2233-5 similar to Saccharomyces cerevisiae C. elegans SCD6 protein
534 2233-22 cDNA EST yk486b9.3 C. elegans 535 2233-93 CDC27Dm D.
melanogaster 536 2084-34 Immune suppressor/V-ATPase 115 kDa Mus
musculus subunit 537 2086-30 V-ATPase A-subunit Aedes aegypti 538
2087-45 H+ATPase Drosophila melanogaster 539 2088-55
40-kDa-V-ATPase subunit Manduca sexta 540 2088-62 vacuolar ATPase
subunit A Drosophila melanogaster 541 2091-26 proton-ATPase-like
protein Homo sapiens 542 2091-31 vacuolar ATPase subunit A
Drosophila melanogaster 543 2092-20 vacuolar ATPase 115 kDa subunit
Homo sapiens 544 2095-18 similar to S. cereviciae vacuolar
H(+)-ATPase C. elegans 54 kD subunit 545 2095-54 H (+)-transporting
ATPase subunit B Manduca sexta 546 2108-8 similar to S. cereviciae
54 kDa V-ATPase C. elegans subunit 547 2154-36 V-ATPase subunit E
Drosophila melanogaster 548 2154-76 V-ATPase subunit A (new
fragment) Aedes aegypti 549 2166-32 V-ATPase C subunit Drosophila
melanogaster 550 2166-33 vacuolar (V-type) H(+)-ATPase B subunit
Helicoverpa virescens 551 2166-90 beta subunit of ATPase Schizaphis
graminum 552 2161-5 ATPase I Plasmodium falciparum 553 2171-24
similar to V-ATPase 116 kd subunit C. elegans 554 2169-82 V-ATPase
subunit E Drosophila melanogaster 555 2187-36 V-ATPase membrane
sector associated Homo sapiens protein M8-9 556 2188-91 V-ATPase
subunit A Candida tropicalis 557 2230-88 vacuolar ATPase G subunit
Manduca sexta 558 2232-61 V-ATPase subunit C D. melanogaster 559
2086-52 Penelope transposable element ORF Drosophila virilis 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 Drosophila melanogaster 563
2202-28 Polyprotein Hepatitis virus C 564 2165-95 DNA polymerase
Choristoneura biennis entomopoxvirus 565 2169-81 reverse
transcriptase Drosophila melanogaster 566 2181-36 reverse
transcriptase Anopheles gambiae 1416 2240-4 alpha-L-fucosidase
precursor Homo sapiens 1417 2240-11 estrogen related receptor alpha
Mus musculus 1418 2240-14 NADH: ubiquinone oxidoreductase 51-kD
Homo sapiens subunit 1419 2240-17 peritrophin 1 Anopheles gambiae
1420 2240-19 small GTPase rac1b Homo sapiens 1421 2240-23 Symplekin
Homo sapiens 1422 2240-26 ribosomal protein L30 Bos taurus 1423
2240-28 60S Ribosomal Protein RPL10A Homo sapiens 1424 2240-29
KIN17 protein D. melanogaster 1425 2240-31 eukaryotic initiation
factor 4 gamma Homo sapiens 1426 2240-38 ornithine decarboxylase
antizyme D. melanogaster 1427 2240-44 electron transfer
flavoprotein Rattus norvegicus 1428 2240-53 EST clone C. elegans
1429 2240-55 glutathione reductase family Musca domestica 1430
2240-58 chymotrypsin-like serine protease C. felis 1431 2240-63
Ferritin subunit 1 D. melanogaster 1432 2240-64 vacuolar ATPase
subunit B D. melanogaster 1433 2240-66 chaperonin containing TCP-1
delta Fugu rubripes 1434 2240-70 1-acyl-glycerol-3-phosphate
acyltransferase Zea mays 1435 2240-71 EST clone AL021106 D.
melanogaster 1436 2240-72 376aa long hypothetical dehydrogenase
Pyrococcus horikoshii 1437 2240-77 chymotrypsin-like serine
protease C. felis 1438 2240-80 EST clone C. elegans 1439 2240-83
chymotrypsin-like serine protease C. felis 1440 2240-90 cytochrome
P450 D. melanogaster 1441 2240-93 enhancer-trap-locus-1 Mus
musculus 1442 2240-94 glycerol-3-phosphate dehydrogenase Ceratitis
capitata 1443 2241-3 FS-H precourser Ctenocephalides felis 1444
2241-5 trypsin-like serine protease Ctenocephalides felis 1445
2241-7 myospheroid protein D. melanogaster 1446 2241-10 Sam50 D.
melanogaster 1447 2241-12 NADH dehydrogenase subunit 2 Chorthippus
parallelus 1448 2241-15 putative protein Arabidopsis thaliana 1449
2241-16 contains EGF-like repeats C. elegans 1450 2241-20 Gcap1
gene product Mus musculus 1451 2241-25 Na(+)-dependent inorganic
phosphate D. melanogaster cotransporter 1452 2241-31 D4L Variola
virus 1453 2241-36 plenty-of-prolines-101; POP101; SH3-philo- Mus
musculus protein 1454 2241-40 EF-1-alpha D. melanogaster 1455
2241-44 F1-ATP synthase epsilon-subunit Ipomoea batatas 1456
2241-54 ribosomal protein S28 Homo sapiens 1457 2241-55 Y-box
protein D. melanogaster 1458 2241-56 short-chain alcohol
dehydrogenase Homo sapiens 1459 2241-59 contains 3 cysteine rich
repeats C. elegans 1460 2241-60 muscle type phosphofructokinase
Canis familiaris 1461 2241-61 Heat shock protein 82 Mus musculus
1462 2241-65 chymotrypsin-like protease C. felis 1463 2241-66
Oligosaccharyltransferase subunit D. melanogaster 1464 2241-70 EST
clone D. melanogaster 1465 2241-72 failed axon connections protein
D. melanogaster 1466 2241-74 Enolase Hymenolepis diminuta 1467
2241-78 multiple exostosis 2 protein Mus musculus 1468 2241-80
Protein on Ecdysone Puffs D. melanogaster 1469 2241-82 paramyosin
D. melanogaster 1470 2241-83 beta-tubulin Bombyx mori 1471 2241-84
natural killer cell enhancing factor Cypninus carpio 1472 2241-86
similar to MYOTUBULARIN-RELATED Homo sapiens PROTEIN 1473 2241-87
Renin Rattus norvegicus 1474 2241-90 Myophilin Echinococcus
multiocularis 1475 2243-10 alpha-actinin D. melanogater 1476
2243-11 monocarboxylate transporter Homo sapiens 1477 2243-13
yk278a10.3 C. elegans 1478 2243-15 selenium donor protein Homo
sapiens 1479 2243-18 acetyl-CoA synthetase D. melanogater 1480
2243-20 cytochrome P450 CYP12A3 Musca domestica 1481 2243-22 NADH
dehydrogenase subunit 4 Anopheles arabiensis 1482 2243-27
Polyubiquitin Cricetulus griseus 1483 2243-28 Moesin D. melanogater
1484 2243-31 QM protein Bombyx mandarina 1485 2243-32 Sec23 protein
Homo sapiens 1486 2243-37 truncated protein S. cereviciae 1487
2243-38 Projectin D. melanogater 1488 2243-39 Unknown Homo sapiens
1489 2243-41 similar to enoyl-CoA hydratase C. elegans 1490 2243-45
similar to dehydrogenase C. elegans 1491 2243-46 trypsin-like
serine protease C. felis 1492 2243-48 Merlin Rattus norvegicus 1493
2243-52 GTP-specific succinyl-CoA synthetase beta Homo sapiens
subunit 1494 2243-53 sod protein (superoxide dismutase) Drosophila
virilis 1495 2243-54 trypsin-like serine protease C. felis 1496
2243-61 chymotrypsin-like serine protease C. felis 1497 2243-66 Tag
B Dictyostelium discoideum 1498 2243-67 hypothetical protien
Arabidopsis thaliana 1499 2243-68 heat shock cognate protein 70
Trichoplusia ni 1500 2243-72 TRIP-1 homologue D. melanogater 1501
2243-73 cytosolic NADP-dependent isocitrate Microtis mexicanis
dehydrogenase 1502 2243-86 progesterone-induced protein Oryctolagus
cuniculus 1503 2243-87 Bmsqd-2 Bombyx mori 1504 2243-91
sodium/iodide symporter Homo sapiens 1505 2243-92 ORF2 Acidianus
ambivalens 1506 2243-94 lysosomal beta-galactosidase Felis cattus
1507 2244-12 tropomyosin isoform 127 D. melanogaster 1508 2244-19
KIAA0181 Homo sapiens 1509 2244-23 plasma membrane calcium ATPase
isoform 1 Homo sapiens 1510 2244-29 NADH dehydrogenase Bos taurus
1511 2244-44 glutamate dehydrogenase D. melanogaster 1512 2244-54
spliceosomal protein D. melanogaster 1513 2244-59 ciliary body
glutathione peroxidase Bos taurus 1514 2244-61
pyridoxal-phoshate-dependent C. elegans aminotransferases 1515
2244-64 Unknown Rattus norvegicus 1516 2244-69 trypsin-like serine
protease C. felis 1517 2244-71 peritrophin 1 Anopheles gambiae 1518
2244-75 NADH dehydrogenase subunit 5 Anopheles gambiae 1519 2244-84
microsomal epoxide hydrolase Rattus norvegicus 1520 2244-86 C54G7.2
gene product C. elegans 1521 2244-91 Aminopeptidase N Plutella
xylostella 1522 2253-2 cytochrome C oxidase H. sapiens 1523 2253-13
Initiation factor 5A Gallus gallus 1524 2253-14 protein phosphatase
type 2A catalytic subunit Bos taurus 1525 2253-16 myosin light
chain 2 D. melanogaster 1526 2253-18 cDNA EST yk462d1.5 C. elegans
1527 2253-19 ribosomal protein S10 H. sapiens 1528 2253-24
aspartyl(asparaginyl)beta-hydroxylase, HAAH H. sapiens 1529 2253-27
larval and adult myosin heavy chain D. melanogaster 1530 2253-33
nervous system antigen 2 D. melanogaster 1531 2253-36 dJ366N23.2 H.
sapiens 1532 2253-40 hrp48.1 D. melanogaster 1533 2253-42 ZnT-1 Mus
musculus 1534 2253-43 aminopeptidase N Manduca sexta 1535 2253-56
Profilin D. melanogaster 1536 2253-59 T26A5. H. sapiens 1537
2253-68 NADH-ubiquinone oxidoreductase 42 kDa D. melanogaster
subunit 1538 2253-78 glycine-rich protein 1539 2253-81
5'-nucleotidase H. sapiens 1540 2253-86 glutathione S-transferase
Anopheles gambiae 1541 2253-87 ferritin subunit 1 D. melanogaster
1542 2253-92 myosin light chain 2 D. melanogaster 1543 2253-94
xylose-proton symport E. coli 1544 2254-4 mature-parasite-infected
erythrocyte surface P. falciparum antigen 1545 2254-6 Fo-ATP
synthase subunit b D. melanogaster 1546 2254-13 similar to
Arabidopsis thaliana male sterility C. elegans protein 2 1547
2254-17 CLN3 protein H. sapiens 1548 2254-21 YbgG B. subtilis 1549
2254-25 peroxisomal protein Synechocystis sp 1550 2254-27
Glutaminase Rattus norvegicus 1551 2254-30 tartan protein D.
melanogaster 1552 2254-33 leucine zipper-EF-hand containing H.
sapiens transmembrane protein 1 1553 2254-39 similar to helicase C.
elegans 1554 2254-43 muscle myosin heavy chain D. melanogaster 1555
2254-45 putative nicotinate phosphoribosyltransferase N. tabacum
1556 2254-51 60S ribosomal protein Mus musculus 1557 2254-54 small
nuclear riboprotein Sm-D H. sapiens 1558 2254-55 nucleoside
diphosphate kinase Salmo salar 1559 2254-60 serine protease C.
felis 1560 2254-63 myospheroid protein D. melanogaster 1561 2254-65
Carboxylesterase Anisopteromalus calandrae 1562 2254-66 siah
binding protein 1 H. sapiens 1563 2254-70 vacuolar ATPase, subunit
M9.7 Manduca sexta 1564 2254-83 Fumarylacetoacetate hydrolase
Rattus norvegicus 1565 2254-84 metalloproteinase 1 Hydra vulgaris
1566 2254-88 alpha-spectrin D. melanogaster 1567 2254-93 NADH
dehydrogenase subunit 6 D. melanogaster 1568 2254-96 cyclophilin
isoform 5 C. elegans 1569 2255-5 similar to mitochondrial ATPase
inhibitors C. elegans 1570 2255-8 yk391f12.5 C. elegans 1571
2255-12 Unknown H. sapiens 1572 2255-17 ribonucleotide reductase
subunit M1 M. musculus 1573 2255-19 docking protein H. sapiens 1574
2255-22 Similar to rat 5E5 antigen H. sapiens 1575 2255-23
ribosomal protein S31 D. melanogaster 1576 2255-25 Similar to
acyl-CoA dehydrogenase C. elegans 1577 2255-28 Arginine tyrosine
kinase H. sapiens 1578 2255-32 ribosomal protein L7a D.,
melanogaster 1579 2255-33 chS-Rex-s G. gallus 1580 2255-39
Phosphoacetylglucosamine mutase C. elegans 1581 2255-41 NADH
dehydrogenase subunit 6 D. melanogaster 1582 2255-45 tRNA-glutamine
synthetase C. elegans 1583 2255-46 p68 M. musculus 1584 2255-49
ABC8 M. musculus 1585 2255-50 kynurenine aminotransferase R. rattus
1586 2255-51 SmD homolog {Gly-Arg repeat} M. musculus 1587 2255-56
epoxide hydrolase S. scrofa 1588 2255-60 Sec23 protein H. sapiens
1589 2255-62 HMG CoA synthase M. musculus 1590 2255-63 dipeptidyl
aminopeptidase-like protein 6 M. musculus 1591 2255-66 retinal rod
Na+/Ca+, K+ exchanger H. sapiens 1592 2255-67 4-hydroxybutyrate
coenzyme A transferase C. elegans 1593 2255-70 hD54 + ins2 isofarm
H. sapiens 1594 2255-73 chromaffin granule ATPase II homolog M.
musculus 1595 2255-77 40S ribosomal protein S10 H. sapiens 1596
2255-79 34/67 kD laminin binding protein S. purpuratus 1597 2255-82
RNA-binding protein lark D., melanogaster 1598 2255-86
thiol-specific antioxidant protein R. norvegicus 1599 2256-7
Similar to Human estrogen-responsive finger H. sapiens protein 1600
2256-11 Trypsin C. felis 1601 2256-12 CEV14 H. sapiens 1602 2256-16
AL021475 C. elegans 1603 2256-21 Heterogenous Nuclear
Ribonucleoprotein C1 H. sapiens 1604 2256-22 b4 integrin interactor
H. sapiens 1605 2256-28 testis enhanced gene transcript protein H.
sapiens 1606 2256-31 synaptic vesicle protein 2B R. norvegicus 1607
2256-40 TNF-alpha stimulated ABC protein H. sapiens 1608 2256-42
carboxypeptidase A H. armigera 1609 2256-46 pherophorin S V.
carteri 1610 2256-52 Fo-ATP synthase subunit b D. melanogaster 1611
2256-54 PDGF associated protein H. sapiens 1612 2256-58 S20
ribosomal protein D. melanogaster 1613 2256-64 ribosomal protein S9
H. sapiens 1614 2256-69 elongation factor 1-gamma Artemia sp 1615
2256-70 conserved hypothetical protein S. pombe 1616 2256-72
fructose 1,6 bisphosphate-aldolase 4C D. melanogaster 1617 2256-73
troponin-T D. melanogaster 1618 2256-80 SRP14 C. familiaris 1619
2256-82 succinyl-CoA synthetase alpha subunit S. scrofa 1620
2256-89 Csa-19 H. sapiens 1621 2256-92 Sacm21 M. musculus 1622
2256-94 apoptosis inhibitor Cydia pomonella granulosis virus 1623
2256-96 ribosomal protein L22 D. melanogaster
[0067] Table III represents a variety of flea HNC nucleic acid
molecules of the present invention.
3TABLE 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.
4TABLE 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
C. felis nucleic acid sequence of Table I, Table II, Table III
and/or Table IV or a complement thereof. For example, an allelic
variant of a C. felis ALN gene including SEQ ID NO:1 is a gene that
occurs at essentially the same locus (or loci) in the genome as the
gene including SEQ ID NO:1, but which, due to natural variations
caused by, for example, mutation or recombination, has a similar
but not identical sequence. Because natural selection typically
selects against alterations that affect function, allelic variants
(i.e. alleles corresponding to, or of, cited nucleic acid
sequences) usually encode proteins having similar activity to that
of the protein encoded by the gene to which they are being
compared. Allelic variants of genes or nucleic acid molecules can
also comprise alterations in the 5' or 3' untranslated regions of
the gene (e.g., in regulatory control regions), or can involve
alternative splicing of a nascent transcript, thereby bringing
alternative exons into juxtaposition. Allelic variants are well
known to those skilled in the art and would be expected to occur
naturally within a given flea such as C. felis, since the genome is
diploid, and sexual reproduction will result in the reassortment of
alleles. For example, SEQ ID NO:162 is apparently an allelic
variant or multiple gene of SEQ ID NO:153.
[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, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Labs Press, and Meinkoth, et
al., 1984, Anal. Biochem. 138, 267-284, each of which is
incorporated by reference herein in its entirety. As explained in
detail in the cited references, the determination of hybridization
conditions involves the manipulation of a set of variables
including the ionic strength (M, in moles/liter), the hybridization
temperature (.degree. C.), the concentration of nucleic acid helix
destabilizing agents (such as formamide), the average length of the
shortest hybrid duplex (n), and the percent G+C composition of the
fragment to which an unknown nucleic acid molecule is being
hybridized. For nucleic acid molecules of at least about 150
nucleotides, these variables are inserted into a standard
mathematical formula to calculate the melting temperature, or
T.sub.m, of a given nucleic acid molecule. As defined in the
formula below, T.sub.m is the temperature at which two
complementary nucleic acid molecule strands will disassociate,
assuming 100% complementarity between the two strands:
T.sub.m=81.5.degree. C.+16.6 log
M+0.41(%G+C)-500/n-0.61(%formamide).
[0073] For nucleic acid molecules smaller than about 50
nucleotides, hybrid stability is defined by the dissociation
temperature (T.sub.d), which is defined as the temperature at which
50% of the duplexes dissociate. For these smaller molecules, the
stability at a standard ionic strength is defined by the following
equation:
T.sub.d=4(G+C)+2(A+T).
[0074] A temperature of 5.degree. C. below T.sub.d is used to
detect hybridization between perfectly matched molecules.
[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.sub.m decreases about 1.degree. C.
for each 1% of mismatched base pairs for hybrids greater than about
150 bp, and Td decreases about 5.degree. C. for each mismatched
base pair for hybrids below about 50 bp. Conditions for hybrids
between about 50 and about 150 base pairs can be determined
empirically and without undue experimentation using standard
laboratory procedures well known to those skilled in the art. These
simple procedures allow one skilled in the art to set the
hybridization conditions (by altering, for example, the salt
concentration, the formamide concentration or the temperature) so
that only nucleic acid hybrids with greater than a specified % base
pair mismatch will hybridize. Stringent hybridization conditions
are commonly understood by those skilled in the art to be those
experimental conditions that will allow less than or equal to about
30% base pair mismatch (i.e., at least about 70% identity). Because
one skilled in the art can easily determine whether a given nucleic
acid molecule to be tested is less than or greater than about 50
nucleotides, and can therefore choose the appropriate formula for
determining hybridization conditions, he or she can determine
whether the nucleic acid molecule will hybridize with a given gene
under stringent hybridization conditions and similarly whether the
nucleic acid molecule will hybridize under conditions designed to
allow a desired amount of base pair mismatch.
[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.times. SSC and 0% formamide, at a
temperature of about 37.degree. 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.times. 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.times. SSC and 0% formamide, the T.sub.m of perfect
hybrids would be about 77.degree. C.:
81.5.degree. C.+16.6
log(0.15M)+(0.41.times.0.37)-(500/150)-(0.61.times.0)-
=77.5.degree. C.
[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.degree.
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.sub.m for a hybridization reaction allowing up to 30% base pair
mismatch will not vary significantly from 47.5.degree. C.
[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.TM.),
Madison, Wis., DNAsis.TM., available from Hitachi Software, San
Bruno, Calif., and MacVector.TM., 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.times.SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising IX SSC and
0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising IX SSC and
0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature of about
37.degree. C. and (b) washing in a solution comprising 1.times. SSC
and 0% formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide, at a temperature
of about 37.degree. C. and (b) washing in a solution comprising
1.times. SSC and 0% formamide, at a temperature of about
47.5.degree. 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.sub.2057, nCfALN.sub.1152, nCfCBP.sub.1128,
nCfCBP.sub.816, nCfNKAB.sub.1714, nCfNKAB.sub.978,
nCfLGIC.sub.2240, nCfLGIC.sub.1707, nCfANON.sub.1429,
nCfANON.sub.1194, nCfMALV.sub.765, nCfMALV.sub.762, nCfOSD.sub.604,
nCfOSD.sub.405, nCfNMDA.sub.1227, nCfNMDA.sub.738,
nCfCLBP1A.sub.633, nCfCLBP1A.sub.441, nCfCLBP2A.sub.631,
nCfCLBP2A.sub.441, nCfLGIC.sub.2739, nCfLGIC.sub.2016,
nCfNAH.sub.2080, nCfNAH.sub.1824, nCfCLIC.sub.2283,
nCfCLIC.sub.786, nCfPL2.sub.1291, nCfPL2.sub.1173, nCfPL3.sub.406,
nCfPL3.sub.243, nCfPL4.sub.974, nCfPL4.sub.1043, nCfPL4.sub.1062,
nCfPL4.sub.855, nCfSVP.sub.1875, nCfSVP.sub.1590, nCfGCC.sub.381,
nCfVGCC.sub.2191, nCfVGCC.sub.1968, nCfVGCC.sub.673,
nCfVGCC.sub.3126, nCfVGCC.sub.2553, nCfAUP.sub.1181,
nCfAUP.sub.306, nCf7B2.sub.2161, nCf7B2.sub.801, nCf7B2.sub.741 or
allelic variants of any of these nucleic acid molecules. Another
preferred isolated protein is encoded by a nucleic acid molecule
having nucleic acid sequence 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, SEQID 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; or
a protein encoded by an allelic variant of any of these listed
nucleic acid molecules.
[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.sub.384, PCfCBP.sub.272,
PCfNKAB.sub.326, PCfLGIC.sub.569, PCfANON.sub.398, PCfMALV.sub.254,
PCfOSD.sub.135, PCfNMDA.sub.246, PCfCLBP1A.sub.147 or
PCfCLBP2A.sub.147. Additionally preferred are proteins encoded by
allelic variants of a nucleic acid molecules encoding proteins
PCfALN.sub.384, PCfCBP.sub.272, PCfNKAB.sub.326, PCfLGIC.sub.569,
PCfANON.sub.398, PCfMALV.sub.254, PCfOSD.sub.135, PCfNMDA.sub.246,
PCfCLBP1A.sub.147, PCfCLBP2A.sub.147, PCfLGIC.sub.672,
PCfNAH.sub.608, PCfCLIC.sub.262, PCfPL2.sub.391, PCfPL3.sub.81,
PCfPL4.sub.285, PCfSVP.sub.530, PCfVGCC.sub.851, PCfAUP.sub.102,
PCf7B2.sub.267, PCf7B2.sub.247. Also preferred are fragments
thereof having at least about 6 amino acid residues.
[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, C. felis HMT and
HNC proteins comprise 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 (including, but not limited to, the proteins consisting
of 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,
fusion proteins and multivalent proteins), and proteins encoded by
allelic variants of nucleic acid molecules encoding proteins having
amino acid sequence SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ IID
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.
[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.sub.2057, nCfALN.sub.1152,
nCfCBP.sub.1128, nCfCBP.sub.816, nCfNKAB.sub.1714, nCfNKAB.sub.978,
nCfLGIC.sub.2240, nCfLGIC.sub.1707, nCfANON.sub.1429,
nCfANON.sub.1194, nCfMALV.sub.765, nCfMALV.sub.762, nCfOSD.sub.604,
nCfOSD.sub.405, nCfNMDA.sub.1227, nCfNMDA.sub.738,
nCfCLBP1A.sub.633, nCfCLBP1A.sub.441, nCfCLBP2A.sub.631,
nCfCLBP2A.sub.441, nCfLGIC.sub.2739, nCfLGIC.sub.2016,
nCfNAH.sub.2080, nCfNAH.sub.1824, nCfCLIC.sub.2283,
nCfCLIC.sub.786, nCfPL2.sub.1291, nCfPL2.sub.1173, nCfPL3.sub.406,
nCfPL3.sub.243, nCfPL4.sub.974, nCfPL4.sub.1043, nCfPL4.sub.1062,
nCfPL4.sub.855, nCfSVP.sub.1875, nCfSVP.sub.1590, nCfVGCC.sub.381,
nCfVGCC.sub.2191, nCfVGCC.sub.1968, nCfVGCC.sub.673,
nCfVGCC.sub.3126, nCfVGCC.sub.2553, nCfAUP.sub.1181,
nCfAUP.sub.306, nCf7B2.sub.2161, nCf7B2.sub.801, nCf7B2.sub.741 as
well as HMT and HNC proteins encoded by allelic variants of such
nucleic acid molecules. A portion of such HMT and HNC nucleic acid
molecule is preferably at least 18 nucleotides in length.
[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.sub.2057, nCfALN.sub.1152,
nCfCBP.sub.1128, nCfCBP816, nCfNKAB.sub.1714, nCfNKAB.sub.978
nCfLGIC.sub.2240, nCfLGIC.sub.1707, nCfANON.sub.1429,
nCfANON.sub.194, nCfMALV.sub.765, nCfMALV.sub.762, nCfOSD.sub.604,
nCfOSD.sub.405, nCfNMDA.sub.1227, nCfNMDA.sub.738,
nCfCLBP1A.sub.633, nCfCLBP1A.sub.441, nCfCLBP2A.sub.631,
nCfCLBP2A.sub.441, nCfLGIC2.sub.739, nCfLGIC.sub.2016,
nCfNAH.sub.2080, nCfNAH.sub.1824, nCfCLIC.sub.2283,
nCfCLIC.sub.786, nCfPL2.sub.1291, nCfPL2.sub.1173, nCfPL3.sub.406,
nCfPL3.sub.243, nCfPL4.sub.974, nCfPL4.sub.1043, nCfPL4.sub.1062,
nCfPL4.sub.855, nCfSVP.sub.1875, nCfSVP.sub.1590, nCfVGCC.sub.381,
nCfVGCC.sub.2191, nCfVGCC.sub.1968, nCfVGCC.sub.673,
nCfVGCC.sub.3126, nCfVGCC.sub.2553, nCfAUP.sub.1181,
nCfAUP.sub.306, nCf7B2.sub.2161, nCf7B2.sub.801, nCf7B2.sub.741 or
by an allelic variant of any of these nucleic acid molecules. In
yet another embodiment, preferred flea HMT and HNC proteins of the
present invention are encoded by nucleic acid molecules comprising
apparently full-length HMT or HNC coding regions respectively,
i.e., nucleic acid molecules encoding an apparently full-length HMT
or HNC proteins.
[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 Ctenocephalides canis,
Ctenocephalides felis, Diamanus montanus, Echidnophaga gallinacea,
Nosopsyllus faciatus, Pulex irritans, Pulex simulans, Tunga
penetrans and Xenopsylla cheopis being more preferred, with C.
felis being even more preferred. Such fleas are also preferred for
the isolation of proteins or nucleic acid molecules of the present
invention.
[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 .beta.-galactosidase, a strep tag
peptide, a T7 tag peptide, a Flag.TM. 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 C. felis HMT and/or HNC nucleic
acid molecules.
[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.times. SSC and 0%
formamide, at a temperature of about 47.5.degree. 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.times. SSC and 0% formamide,
at a temperature of about 47.5.degree. 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.TM.
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.sub.2057, nCfALN.sub.1152, nCfCBP.sub.1128, nCfCBP.sub.816,
nCfNKAB.sub.1714, nCfNKAB.sub.978 nCfLGIC.sub.2240,
nCfLGIC.sub.1707, nCfANON.sub.1429, nCfANON.sub.1194,
nCfMALV.sub.765, nCfMALV.sub.762, nCfOSD.sub.604, nCfOSD.sub.405,
nCfNMDA.sub.1227, nCfNMDA.sub.738, nCfCLBP1A.sub.633,
nCfCLBP1A.sub.441, nCfCLBP2A.sub.631, nCfCLBP2A.sub.441,
nCfLGIC.sub.2739, nCfLGIC.sub.2016, nCfNAH.sub.2080,
nCfNAH.sub.1824, nCfCLIC.sub.2283, nCfCLIC.sub.786,
nCfPL2.sub.1291, nCfPL2.sub.1173, nCfPL3.sub.406, nCfPL3.sub.243,
nCfPL4.sub.974, nCfPL4.sub.1043, nCfPL4.sub.1062, nCfPL4.sub.855,
nCfSVP.sub.1875, nCfSVP.sub.1590, nCfVGCC.sub.381,
nCfLGIC.sub.1291, nCfVGCC.sub.1968, nCfVGCC.sub.673,
nCfVGCC.sub.3126, nCfVGCC.sub.2553, nCfAUP.sub.1181,
nCfAUP.sub.306, nCf7B2.sub.2161, nCf7B2.sub.801, nCf7B2.sub.741 or
allelic variants of these nucleic acid molecules. Another preferred
nucleic acid molecule of the present invention includes at least a
portion of nucleic acid sequence 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 molecule of Table I, Table II, Table III or
Table IV, as well as allelic variants of nucleic acid molecules
having these nucleic acid sequences and homologues of nucleic acid
molecules having these nucleic acid sequences; preferably such a
homologue encodes or is complementary to a nucleic acid molecule
that encodes at least one epitope that elicits an immune response
against a protein having an 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. Such nucleic acid molecules can
include nucleotides in addition to those included in the SEQ ID
NOs, such as, but not limited to, a full-length gene, a full-length
coding region, a nucleic acid molecule encoding a fusion protein,
or a nucleic acid molecule encoding a multivalent protective
compound.
[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.sub.384, PCfCBP.sub.272,
PCfNKAB.sub.326, PCfLGIC.sub.569, PCfANON.sub.398, PCfMALV.sub.254,
PCfOSD.sub.135, PCfNMDA.sub.246, PCfCLBP1A.sub.147,
PCfCLBP2A.sub.147, PCfLGIC.sub.672, PCfNAH.sub.608,
PCfCLIC.sub.262, PCfPL2.sub.391, PCfPL3.sub.81, PCfPL4.sub.285,
PCfSVP.sub.530, PCfVGCC.sub.851, PCfAUP.sub.102, PCf7B2.sub.267,
PCf7B2.sub.247 and/or a protein encoded by a nucleic acid molecule
having a sequence of Table I, Table II, Table III and/or Table
IV.
[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.sup.st instar larvae;
3.sup.rd instar larvae, wandering larvae, prepupal larvae, pupae
and whole adult flea cDNA libraries as well as genomic DNA
libraries. Similarly, preferred DNA sources to screen or from which
to amplify nucleic acid molecules include flea prepupal cDNA, adult
cDNA and genomic DNA. Techniques to clone and amplify genes are
disclosed, for example, in Sambrook et al., ibid.
[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 C. felis HMT and/or HNC nucleic acid
molecules or other flea HMT and/or HNC nucleic acid molecules.
Oligonucleotides of the present invention can be RNA, DNA, or
derivatives of either. The minimum size of such oligonucleotides is
the size required for formation of a stable hybrid between an
oligonucleotide and a complementary sequence on a nucleic acid
molecule of the present invention. A preferred oligonucleotide of
the present invention has a maximum size of preferably about 100 to
200 nucleotides. The present invention includes oligonucleotides
that can be used as, for example, probes to identify nucleic acid
molecules, primers to produce nucleic acid molecules, or
therapeutic reagents to inhibit flea HMT and/or HNC protein
production or activity (e.g., as antisense-, triplex formation-,
ribozyme- and/or RNA drug-based reagents). The present invention
also includes the use of such oligonucleotides to protect animals
from disease using one or more of such technologies. Appropriate
oligonucleotide-containing therapeutic compositions can be
administered to an animal using techniques known to those skilled
in the art.
[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.sub.L and lambda p.sub.R and
fusions that include such promoters), bacteriophage T7, T7lac,
bacteriophage T3, bacteriophage SP6, bacteriophage SP01,
metallothionein, alpha-mating factor, Pichia alcohol oxidase,
alphavirus subgenomic promoter, antibiotic resistance gene,
baculovirus, Heliothis zea insect virus, vaccinia virus,
herpesvirus, raccoon poxvirus, other poxvirus, adenovirus,
cytomegalovirus (such as immediate early promoter), simian virus
40, retrovirus, actin, retroviral long terminal repeat, Rous
sarcoma virus, heat shock, phosphate and nitrate transcription
control sequences as well as other sequences capable of controlling
gene expression in prokaryotic or eukaryotic cells. Additional
suitable transcription control sequences include tissue-specific
promoters and enhancers as well as lymphokine-inducible promoters
(e.g., promoters inducible by interferons or interleukins).
Transcription control sequences of the present invention can also
include naturally occurring transcription control sequences
naturally associated with fleas, such as C. felis transcription
control sequences.
[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.sub.205.sub.7, nCfALN.sub.1152,
nCfCBP.sub.1128, nCfCBP.sub.816, nCfNKAB.sub.1714, nCfNKAB.sub.978
nCfLGIC.sub.2240, nCfLGIC.sub.1707, nCfANON.sub.1429,
nCfANON.sub.1194, nCfMALV.sub.765, nCfMALV.sub.762, nCfOSD.sub.604,
nCfOSD.sub.405, nCfNMDA.sub.1227, nCfMDA.sub.738,
nCfCLBP1A.sub.633, nCfCLBP1A.sub.441, nCfCLBP2A.sub.631,
nCfCLBP2A.sub.441, nCfLGIC.sub.2739, nCfLGIC.sub.2016,
nCfNAH.sub.2080, nCfNAH.sub.1824, nCfCLIC.sub.2283,
nCfCLIC.sub.786, nCfPL2.sub.1291, nCfPL2.sub.1173, nCfPL3.sub.406,
nCfPL3.sub.243, nCfPL4.sub.974, nCfPL4.sub.1043, nCfPL4.sub.1062,
nCfPL4.sub.855, nCfSVP.sub.1875, nCfSVP.sub.1590, nCfVGCC.sub.381,
nCfVGCC.sub.2191, nCfGCC.sub.1968, nCfVGCC.sub.673,
nCfVGCC.sub.3126, nCfVGCC.sub.2553, nCfAUP.sub.1181,
nCfAUP.sub.306, nCf7B2.sub.2161, nCf7B2.sub.801,
nCf7B2.sub.741.
[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 C. felis HMT and HNC nucleic acid molecules disclosed
herein. Preferred nucleic acid molecules with which to transform a
cell include nucleic acid molecules having a sequence of Table I,
Table II, Table III and/or Table IV. Particularly preferred nucleic
acid molecules with which to transform a cell include
nCfALN.sub.2057, nCfALN.sub.1152, nCfCBP.sub.1128, nCfCBP.sub.816,
nCfNKAB.sub.1714, nCfNKAB.sub.978 nCfLGIC.sub.2240,
nCfLGIC.sub.1707, nCfANON.sub.1429, nCfANON.sub.1194,
nCfMALV.sub.765, nCfMALV.sub.762, nCfOSD.sub.604, nCfOSD.sub.405,
nCfNMDA.sub.1227, nCfNMDA.sub.738, nCfCLBP1A.sub.633,
nCfCLBP1A.sub.441, nCfCLBP2A.sub.631, nCfCLBP2A.sub.441,
nCfLGIC.sub.2739, nCfLGIC.sub.2016, nCfNAH.sub.2080,
nCfNAH.sub.1824, nCfCLIC.sub.2283, nCfCLIC.sub.786,
nCfPL2.sub.1291, nCfPL2173, nCfPL3.sub.406, nCfPL3243,
nCfPL4.sub.974, nCfPL4.sub.1043, nCfPL4.sub.1062, nCfPL4.sub.8551
nCfSVP.sub.1875, nCfSVP.sub.1590, nCfVGCC.sub.381,
nCfVGCC.sub.2191, nCfVGCC.sub.1968, nCfVGCC.sub.673,
nCfVGCC.sub.3126, nCfVGCC.sub.2553, nCfAUP.sub.1181,
nCfAUP.sub.306, nCf7B2.sub.2161, nCf7B2.sub.801, or
nCf7B2.sub.741.
[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
Escherichia coli, including E. coli K-12 derivatives; Salmonella
typhi; Salmonella typhimurium, including attenuated strains such as
UK-1 .sub..chi.3987 and SR-11 .sub..chi.4072; Caulobacter; Pichia;
Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFK
cells; CV-1 cells; COS cells; Vero cells; and non-tumorigenic mouse
myoblast G8 cells (e.g., ATCC CRL 1246). Additional appropriate
mammalian cell hosts include other kidney cell lines, other
fibroblast cell lines (e.g., human, murine or chicken embryo
fibroblast cell lines), myeloma cell lines, Chinese hamster ovary
cells, mouse NIH/3T3 cells, LMTK.sup.31 cells and/or HeLa cells. In
one embodiment, the proteins may be expressed as heterologous
proteins in myeloma cell lines employing immunoglobulin
promoters.
[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 E. coli; or be retained on the
outer surface of a cell or viral membrane.
[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-C. felis HMT or HNC antibodies). As used
herein, the term "selectively binds to" an HMT and/or HNC protein
refers to the ability of antibodies of the present invention to
preferentially bind to specified proteins and mimetopes thereof of
the present invention. Binding can be measured using a variety of
methods standard in the art including enzyme immunoassays (e.g.,
ELISA), immunoblot assays, etc.; see, for example, Sambrook et al.,
ibid., and Harlow, et al., 1988, Antibodies, a Laboratory Manual,
Cold Spring Harbor Labs Press; Harlow et al., ibid., is
incorporated by reference herein in its entirety. An anti-HMT or
anti-HNC antibody of the present invention preferably selectively
binds to a flea HMT or HNC protein respectively in such a way as to
inhibit the function of that protein.
[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.TM. adjuvant (Vaxcel.TM., 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 .mu.g 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 .mu.g, 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.times.10.sup.4 to about 1.times.10.sup.8 virus plaque forming
units (pfa) per kilogram body weight of the animal. Administration
protocols are similar to those described herein for protein-based
vaccines, with subcutaneous, intramuscular, intranasal,
intraocular, conjunctival, and oral administration routes being
preferred.
[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, E. coli, Listeria,
Mycobacterium, S. frugiperda, yeast, (including Saccharomyces
cerevisiae and Pichia pastoris), BHK, CV-1, myoblast G8, COS (e.g.,
COS-7), Vero, MDCK and CRFK recombinant cells. Recombinant cell
vaccines of the present invention can be administered in a variety
of ways but have the advantage that they can be administered
orally, preferably at doses ranging from about 10.sup.8 to about
10.sup.12 cells per kilogram body weight. Administration protocols
are similar to those described herein for protein-based vaccines.
Recombinant cell vaccines can comprise whole cells, cells stripped
of cell walls or cell lysates.
[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 C. felis HMT and/or HNC protein of the
present invention, 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 (b) determining if the
putative inhibitory compound inhibits the activity. HMT and/or HNC
protein activity can be determined in a variety of ways known in
the art, including but not limited to determining the ability of
HMT and/or HNC protein to bind to or otherwise interact with a
substrate. Such conditions under which a HMT and/or HNC protein has
HMT and/or HNC protein activity include conditions in which a HMT
and/or HNC protein has a correct three-dimensionally folded
structure under physiologic conditions, i.e. physiologic pH,
physiologic ionic concentrations, and physiologic temperatures.
[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 Ctenocephalides felis and the use
of isolated RNA to construct subtracted and unsubtracted cDNA
libraries.
[0235] Approximately 10,000 hindguts and Malpighian tubules were
dissected from equal numbers of cat blood fed and unfed adult C.
felis with a male to female ratio of 1 to 4, and total RNA was
extracted using a guanidine isothiocyanate lysis buffer and the
standard procedure described by Sambrook et al. Poly-A enriched
mRNA was purified from total RNA above using a mRNA Purification
Kit, available from Pharmacia Biotech, Piscataway, N.J., following
the manufacturer's protocol. The same procedures were used to
extract total RNA and isolate poly-A enriched mRNA from the
dissected C. felis bodies following removal of HMT, referred to
hereinafter as "non-HMT mRNA".
[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.TM. 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 (.mu.g) of HMT mRNA
was used as the template for synthesis of the tester material and 2
.mu.g 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.RTM.2.1 vector, available from Invitrogen, Carlsbad,
Calif., using an Original TA Cloning.RTM. Kit, available from
Invitrogen. The ligation reaction was then used to transform
INV.alpha.F' One Shot.TM. 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 .beta.-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.TM. 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.TM. Dye Terminator Cycle Sequencing Ready Reaction
Kit or the PRISM.TM. dRhodamine Terminator Cycle Sequencing Ready
Reaction Kit or the PRISM.TM. BigDye.TM. 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.TM. 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 C. felis with a male to female
ratio of 1:4. Total RNA was extracted using a guanidine
isothiocyanate lysis buffer and procedures described in Sambrook et
al., followed by isolation using a mRNA purification kit, available
from Pharmacia, according to the manufacturer's protocols. The
library was constructed with 5 .mu.g of isolated mRNA using a
ZAP-cDNA.RTM. cDNA synthesis kit, and packaged using a
ZAP-cDNA.RTM. Gigapack.RTM. gold cloning kit, both available from
Stratagene, La Jolla, Calif. The resultant HMT library was
amplified to a titer of about 5.times.10.sup.9 plaque forming units
per milliliter (pfu/ml). Single clone excisions were performed
using the Ex-Assist.TM. helper phage, available from Stratagene,
and used to create double stranded plasmid template for sequencing
using the manufacturer's protocols with the following exceptions.
Following incubation of the SOLR cells with the cleared phage
lysate, the mixture was used to inoculate LB broth, and the mix was
incubated overnight and then subjected to mini-prep plasmid
preparation and sequencing as described for the subtracted HMT
library above.
EXAMPLE 2
[0241] This Example describes the isolation of RNA from the head
and nerve cord (HNC) of Ctenocephalides felis and the use of
isolated RNA to construct subtracted and unsubtracted cDNA
libraries.
[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 C. felis with a male to
female ratio of 1 to 4, and total RNA was extracted using a
guanidine isothiocyanate lysis buffer and the standard procedure
described by Sambrook et al. Approximately 618 .mu.g of total RNA
was recovered. Poly-A enriched mRNA was purified from total RNA
above using a mRNA Purification Kit, available from Pharmacia,
following the manufacturer's protocol. Approximately 13 .mu.g of
mRNA was isolated. The same procedures were used to extract total
RNA and isolate poly-A enriched mRNA from the dissected C. felis
bodies following removal of HNC tissues, referred to hereinafter as
"non-HNC mRNA".
[0243] Suppression subtractive PCR was conducted as described in
Example 1 using a PCR-Select.TM. cDNA Subtraction kit, available
from Clontech, under the following conditions. Two micrograms
(.mu.g) of HNC mRNA was used as the template for synthesis of the
tester material and 2 .mu.g 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.varies..TM. bacteria, available from Gibco-BRL, Gaithersburg,
MD. Portions of the transformation mixes were used to inoculate LB
broth cultures containing 100 .mu.g/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.TM. 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 C.
felis and poly-A RNA was isolated as described above. About seven
.mu.g of HNC poly-A RNA was used to construct a cDNA library using
Stratagene's .lambda.ZAP-cDNA Synthesis Kit and protocol. The
resultant HNC library was amplified to a titer of about
5.times.10.sup.9 plaque forming units per milliliter (pfu/ml).
Single clone excisions were performed using the Ex-Assist helper
phage, available from Stratagene, and used to create double
stranded plasmid template for sequencing using the manufacturer's
protocols with the following exceptions. Following incubation of
the SOLR cells with the cleared phage lysate, the mixture was used
to inoculate LB broth, and the mix was incubated overnight and then
subjected to mini-prep plasmid preparation and sequencing as
described for the subtracted library above.
EXAMPLE 3
[0246] This example describes the production of a C. felis cDNA
pool by Rapid Amplification of cDNA Ends (RACE cDNA pool).
[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.times. 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.degree. C. for 2 hours followed by centrifugation at
10,000.times. 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 .mu.g and the yield from fed fleas was 1500 .mu.g.
[0248] Six-hundred .mu.g 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 .mu.g of mRNA were isolated based on
spectrophotometer analysis and ethidium bromide staining. One .mu.g
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 C. felis allantoinase
nucleic acid molecule of the present invention. This example also
describes the expression of allantoinase mRNA in a variety of flea
tissues.
[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.sub.682. The insert was
isolated by gel purification using a Gel Purification kit,
available from Qiagen, Chatsworth, Calif. Approximately 50
nanograms (ng) of purified nCfALN.sub.682 was used to construct a
.sup.32P .alpha.-dATP labeled DNA probe using a Megaprime DNA
labeling kit, available from Amersham, Arlington Heights, Ill.,
using the manufacturer's protocols.
[0251] The .sup.32P .alpha.-dATP labeled probe was used in a
standard plaque lift hybridization procedure to isolate a clone
from the HMT lambda-ZAP unsubtracted cDNA library described in
Example 1. The following hybridization conditions were used,
hereinafter referred to as "standard hybridization conditions".
Filters were hybridized with about 1.times.10.sup.6 counts per
minute (cpm) per ml of the probe in 5.times. SSPE, (see Sambrook et
al., ibid.), 1.2% sodium dodecyl sulfate (SDS), 0.1 mg/mil salmon
sperm DNA and 5.times. Denhardt's reagent, (see Sambrook et al.,
ibid.), at 55.degree. C. for about 14 hours. The filters were
washed as follows: (a) 10 minutes with 5.times. SSPE and 1% SDS,
(b) 10 minutes with 2.times. SSPE and 1% SDS, (c) 10 minutes with
1.times. SSPE and 0.5% SDS, and (d) 10 minutes with 0.5.times. SSPE
and 1% SDS. All washes were conducted at 55.degree. C. Plaques that
hybridized strongly to the probe were isolated and subjected to in
vivo excision. In vivo excision was performed using the Stratagene
Ex-Assist.TM. helper phage system and protocols, to convert a
positive plaque to pBluescript.TM. plasmid DNA. Sequencing was
conducted using standard sequencing methods following preparation
of DNA with a Qiagen Qiaprep.TM. spin mini prep kit using the
manufacturer's instructions and restriction enzyme digestion with
about 1 .mu.l of 20 U/.mu.l each of EcoRI and XhoI, available from
New England Biolabs, Beverly, Mass. A clone was isolated from a
primary plaque, containing a nucleic acid molecule of about 2057
base pairs, referred to herein as nCfALN.sub.2057, having a
nucleotide sequence denoted herein as SEQ ID NO:1. The complement
of SEQ ID NO:1 is represented herein as SEQ ID NO:3. Sequencing of
nCfALN.sub.682 indicates that nCfALN.sub.682 shared 100% identity
with nucleotides 855 through 1536 of SEQ ID NO:1.
[0252] Translation of SEQ ID NO:1 suggests that nucleic acid
molecule nCfALN.sub.2057 encodes a full-length allantoinase protein
of 384 amino acids, referred to herein as PCfALN.sub.384, having an
amino acid sequence represented by SEQ ID NO:2, assuming the
initiation codon spans from nucleotide 152 through nucleotide 154
of SEQ ID NO:1 and the termination codon spans from nucleotide 1304
through nucleotide 1306 of SEQ ID NO:1. The coding region encoding
PCfALN.sub.384, is represented by nucleic acid molecule
nCfALN.sub.1152, having a coding strand with the nucleic acid
sequence represented by SEQ ID NO:4 and a complementary strand with
nucleic acid sequence represented by SEQ ID NO:6. The amino acid
sequence of PCfALN.sub.384, also represented as SEQ ID NO:5,
predicts that PCfALN.sub.384 has an estimated molecular weight of
about 42.2 kilodaltons (kDa) and an estimated isoelectric point
(pI) of about 6.
[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 Rana
catesbeiana (bullfrog) allantoinase protein, GenBank Accession No.
458126. Comparison of SEQ ID NO:4 with nucleic acid sequences
reported in GenBank indicates that SEQ ID NO:4 showed the most
homology, i.e., about 51% identity, with a Rana catesbeiana nucleic
acid molecule, GenBank Accession number U03471. Percent identity
calculations were performed using GCG version 9.0 using default
parameters.
[0254] The coding region of nCfALN.sub.2057, i.e. SEQ ID NO:4, was
PCR amplified from the pBluescript.TM. clone described above as the
template, using sense primer ALN-FE, having nucleotide sequence 5'
GCG GAT CCT ATG CTG AAT TGC AAG AAC CTT G 3', having a BamHI site
indicated in bold, designated herein as SEQ ID NO:37, and
anti-sense primer ALN-RE, having nucleotide sequence 5.degree. CAG
GTA CCC TCT TTT AGA AGC ACC GGT CCC 3', having a KpnI site
indicated in bold, designated herein as SEQ ID NO:38. PCR reactions
were performed using the following amplification cycles: (a) one
cycle at 95.degree. C. for thirty seconds; (b) thirty cycles at
95.degree. C. for twenty seconds, 50.degree. C. for twenty seconds,
and 72.degree. C. for two minutes; and (c) one cycle at 72.degree.
C. for five minutes, hereinafter referred to as "standard
thermocycling conditions", in reactions containing 2.5 mM
MgCl.sub.2, 0.2 mM dNTPs, 1 .mu.M of each primer, 0.5 .mu.l of
5U/.mu.l Taq polymerase, 1 .mu.l of 1 .mu.g/.mu.l template, and 3
.mu.l of 10.times. Taq buffer, hereinafter referred to as "standard
PCR reaction conditions". 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-nCfALN.sub.1152, was transformed into E.
coli strain BL21, available from Novagen Inc., Madison, Wis., to
form recombinant cell E. coli:pTrc-nCfALN.sub.1152.
[0255] The recombinant cell was grown under standard conditions and
then incubated in the presence of 0.5 .mu.M
isopropylthio-.beta.-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.TM. chelating column charged with NiCl.sub.2, available from
Pharmacia, and was shown to contain the His tag of the vector when
subjected to automated protein sequencing by Edman degradation.
[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
C. felis having a male to female ratio of 1:4. Total RNA was
separately extracted from HMT tissues and the HMT-less carcasses
that resulted from these dissections as follows. The tissues were
frozen at -80.degree. C., ground into a powder with a mortar and
pestle, and the powders were equally divided into four 2-ml
eppendorf tubes each containing 1 ml of lysis buffer. The lysis
buffer contained 4 M guanidinium thiocyanate, 25 mM sodium citrate,
pH 7.0, 3% sarcosyl, 0.5M 2-mercaptoethanol, 0.1% antifoam, and 1
mM aurintricarboxylic acid, all available from Sigma Chemical
Corporation, St. Louis, Mo. After mixing, the tubes were spun at
14,000 rpm for 2 minutes and the supernatants were transferred to
separate 2 ml eppendorf tubes containing 250 .mu.l of phenol,
available from Aldrich, Milwaukee, Wis. After mixing, the tubes
were spun at 14,000 rpm for 5 minutes and the supernatants were
transferred to new 2-ml tubes. This process was repeated 3 times
until no proteinaceous matter was visible at the phenol/lysis
buffer interface, then 250 .mu.l of chloroform was added to each
tube and the contents mixed and spun at 14,000 rpm for 5 minutes
followed by transferring the supernatant to a new tube. A volume of
isopropanol equal to the volume of the supernatant was added to
each tube and the tubes placed on ice for 5 minutes. The tubes were
then spun at 14,000 rpm at room temperature for 15 minutes, the
supernatants were removed and discarded and the remaining RNA
pellets were washed with 70% ethanol and dried. The RNA pellets
were resuspended in 100 .mu.l of TE (10 mM Tris, 1 mM
ethylenediaminetetraacetic acid (EDTA)). The quantity of RNA in
each tube was then determined using a spectrophotometer.
[0257] Approximately 10 .mu.g of each RNA was added to separate
tubes containing 18.75 .mu.l of loading buffer, which consists of
50% formamide, 16% formaldehyde, 17% water, 7% glycerol, 1.times.
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 .mu.l ethidium bromide, and 10 .mu.l
bromophenol blue dye, all available from Sigma. The tubes were
heated to 95.degree. 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.times. 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.times. SSPE
as the transfer buffer onto Nytran.RTM. nylon membrane, available
from Schleicher and Schuell Inc., Keene, N.H. The membrane was UV
cross-linked using the Stratalinker.RTM., available from
Stratagene, then prehybridized at 42.degree. C. in 50% formamide,
5.times. SSPE, 1.2% SDS, 5.times. Denhardt's reagent, 2.5 mM EDTA,
and 100 .mu.g/ml salmon sperm DNA. A probe comprising the
allantoinase EST nucleic acid molecule, nCfALN.sub.682 was labeled
with .alpha.-.sup.32P-ATP using a DNA labeling kit, available from
Amersham and added to the buffer at a concentration of
approximately 1.times.10.sup.6 cpm/ml, and allowed to hybridize for
18 hours at 42.degree. C. The blot was then washed as follows: 10
minutes at 42.degree. C. in 4.times. SSPE and 1% SDS; 10 minutes at
42.degree. C. in 2.times. SSPE and 1% SDS; 10 minutes at 42.degree.
C. with 0.5.times. SSPE and 0.5.times. SDS; and 10 minutes at
42.degree. C. with 0.25.times. SSPE and 0.25% SDS. The blot was
then exposed to film for 1 hour, and the film was developed using
standard procedures. Analysis of the developed film revealed that
allantoinase mRNA was present in HMT tissues but was not present in
non-HMT tissues.
[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
.alpha.-.sup.32P-ATP labeled nCfALN.sub.682 probe as described
above. Analysis of the developed film revealed that allantoinase
mRNA was expressed in all adult fleas tested regardless of feeding
conditions and was expressed by all life stages except for eggs and
pupae, the two life stages which do not feed or excrete urine.
EXAMPLE 5
[0259] This example describes the cloning, sequencing, recombinant
protein expression and purification of a C. felis chitin-binding
protein nucleic acid molecule. This example also describes the
expression of chitin-binding protein mRNA in a variety of flea
tissues.
[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 Bombyx mori (silkworm). This
clone was digested with EcoRI to excise an insert about 429
nucleotides in length, referred to as chitin-binding protein (CBP)
nucleic acid molecule nCfCBP.sub.429. The insert was isolated by
gel purification using a Gel Purification kit, available from
Qiagen. Approximately 50 ng of purified nCfCBP.sub.429 was used to
construct a .sup.32P .alpha.-dATP labeled DNA probe using a
Megaprime DNA labeling kit, available from Amersham, using the
manufacturer's protocols.
[0261] The .sup.32P .alpha.-dATP labeled probe was used in a plaque
lift hybridization procedure to isolate a clone from the HMT
lambda-ZAP unsubtracted cDNA library described in Example 1, using
standard hybridization conditions described in Example 4. Plaques
that hybridized strongly to the probe were isolated and subjected
to in vivo excision. In vivo excision was performed using the
Stratagene Ex-Assist.TM. helper phage system and protocols, to
convert a positive plaque to pBluescript.TM. plasmid DNA, and
sequencing was conducted following preparation of DNA with a Qiagen
Qiaprep.TM. spin mini prep kit using the manufacturer's
instructions and restriction enzyme digestion with about 1 .mu.l of
20 U/.mu.l each of EcoRI and XhoI, available from New England
Biolabs. A clone was isolated from a primary plaque, containing a
nucleic acid molecule of about 1128 base pairs, referred to herein
as nCfCBP.sub.1128, having a nucleotide sequence denoted herein as
SEQ ID NO:7. The complement of SEQ ID NO:7 is represented herein as
SEQ ID NO:9. Sequencing of nCfCBP.sub.429 indicated that
nCfCBP.sub.429 shares 100% identity with nucleotides 148 through
576 of SEQ ID NO:7.
[0262] Translation of SEQ ID NO:7 suggests that nucleic acid
molecule nCfCBP.sub.1128 encodes a full-length chitin-binding
protein of 272 amino acids, referred to herein as PCfCfCBP.sub.272,
having an amino acid sequence represented by SEQ ID NO:8, assuming
the initiation codon spans from nucleotide 6 through nucleotide 8
of SEQ ID NO:7 and the termination codon spans from nucleotide 822
through nucleotide 824 of SEQ ID NO:7. The coding region encoding
PCfCBP.sub.272, is represented by nucleic acid molecule
nCfCBP.sub.816, having a coding strand with the nucleic acid
sequence represented by SEQ ID NO:10 and a complementary strand
with nucleic acid sequence represented by SEQ ID NO:12. The amino
acid sequence of PCfCBP.sub.272, also represented as SEQ ID NO:11,
predicts that PCfCBP.sub.272 has an estimated molecular weight of
about 30.6 kDa and an estimated pI of about 7.3.
[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 Lucilia
cuprina peritrophin-44 protein, GenBank Accession No. 407976.
Comparison of SEQ ID NO:10 with nucleic acid sequences reported in
GenBank indicates that SEQ ID NO:10 showed the most homology, i.e.,
about 40% with a Lucilia cuprina peritrophin-44 nucleic acid
molecule, GenBank Accession number L25106. Percent identity
calculations were performed using GCG version 9.0 using default
parameters.
[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.TM. 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.sub.769, was transformed into E.
coli strain BL21, available from Novagen, to form recombinant cell
E. coli:pTrc-nCfCBP.sub.769. The recombinant cell was grown under
standard conditions and then incubated in the presence of 0.5 .mu.M
IPTG to induce expression of recombinant protein, predicted to be a
protein of approximately 32 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 32-kDa protein. The protein product was purified by liquid
chromatography using a HiTrap.TM. chelating column charged with
NiCl.sub.2, available from Pharmacia, and was shown to contain the
His tag of the vector when subjected to automated protein
sequencing by Edman degradation.
[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
.alpha.-.sup.32P-ATP labeled nCfCBP.sub.429 under the Northern
Blotting conditions described in Example 4. Analysis of the
developed film revealed that CBP mRNA was expressed in HMT tissues
but not in non-HMT tissues. CBP mRNA was also detected in all adult
fleas tested regardless of feeding conditions but was not detected
in any of the non-adult life stages.
EXAMPLE 6
[0266] This example describes the cloning and sequencing of a C.
felis sodium/potassium ATPase, beta subunit nucleic acid
molecule.
[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 Drosophila
melanogaster. This clone was digested with EcoRI to excise an
insert about 439 nucleotides in length, referred to as flea NKB
nucleic acid molecule nCfNKAB.sub.439. The insert was isolated by
gel purification using a Gel Purification kit, available from
Qiagen. Approximately 50 ng of purified nCfNKAB.sub.439 was used to
construct a .sup.32P .alpha.-dATP labeled DNA probe using a
Megaprime DNA labeling kit, available from Amersham, using the
manufacturer's protocols.
[0268] The .sup.32P .alpha.-dATP labeled probe was used in a plaque
lift hybridization procedure to isolate a clone from the HMT
lambda-ZAP unsubtracted cDNA library described in Example 1, using
standard hybridization conditions described in Example 4. Plaques
that hybridized strongly to the probe were isolated and subjected
to in vivo excision. In vivo excision was performed using the
Stratagene Ex-Assist.TM. helper phage system and protocols, to
convert a positive plaque to pBluescript.TM. plasmid DNA, and
sequencing was conducted following preparation of DNA with a Qiagen
Qiaprep.TM. spin mini prep kit using the manufacturer's
instructions and restriction enzyme digestion with about 1 .mu.l of
20 U/.mu.l each of EcoRI and XhoI, available from New England
Biolabs. A clone was isolated from a secondary plaque, containing a
nucleic acid molecule of about 1714 base pairs, referred to herein
as nCfNKAB.sub.1714, having a nucleotide sequence denoted herein as
SEQ ID NO:13. The complement of SEQ ID NO:13 is represented herein
as SEQ ID NO:15. Sequencing of nCfNKAB.sub.439 indicates that
nCfNKAB.sub.439 shared 100% identity with nucleotides 907 through
1345 of SEQ ID NO:13.
[0269] Translation of SEQ ID NO:13 suggests that nucleic acid
molecule nCfNKAB.sub.1714 encodes a full-length NKAB protein of 326
amino acids, referred to herein as PCfNKAB.sub.326, having an amino
acid sequence represented by SEQ ID NO:14, assuming the initiation
codon spans from nucleotide 294 through nucleotide 296 of SEQ ID
NO:13 and the termination codon spans from nucleotide 1272 through
nucleotide 1274 of SEQ ID NO:13. The coding region encoding
PCfNKAB.sub.326 is represented by nucleic acid molecule
nCfNKAB.sub.978, having a coding strand with the nucleic acid
sequence represented by SEQ ID NO:16 and a complementary strand
with nucleic acid sequence represented by SEQ ID NO:18. The amino
acid sequence of PCfNKAB.sub.326, also represented by SEQ ID NO:17
predicts that PCfNKAB.sub.326 has an estimated molecular weight of
about 37.7 kDa and an estimated pI of about 5.
[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
Drosophila melanogaster nervous system antigen 2 protein, GenBank
Accession No. 881344. Comparison of SEQ ID NO:16 with nucleic acid
sequences reported in GenBank indicates that SEQ ID NO:16 showed
the most homology, i.e., about 52% identity, with a Drosophila
melanogaster nervous system antigen 2 nucleic acid molecule,
GenBank Accession number U22440. Percent identity calculations were
performed using GCG version 9.0 using default parameters.
EXAMPLE 7
[0271] This example describes the cloning and sequencing of a C.
felis ligand-gated ion channel nucleic acid molecule. This example
also describes the expression of ligand-gated ion channel mRNA in a
variety of flea tissues.
[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.sub.376. The insert was isolated by gel
purification using a Gel Purification kit, available from Qiagen.
Approximately 50 ng of purified nCfLGIC.sub.376 was used to
construct a .sup.32P .alpha.-dATP labeled DNA probe using a
Megaprime DNA labeling kit available from Amersham, using the
manufacturer's protocols.
[0273] The .sup.32P .alpha.-dATP labeled probe was used in a plaque
lift hybridization procedure to isolate a clone from the HMT
lambda-ZAP unsubtracted cDNA library described in Example 1, using
standard hybridization conditions described in Example 4. Plaques
that hybridized strongly to the probe were isolated and subjected
to in vivo excision. In vivo excision was performed using the
Stratagene Ex-Assist.TM. helper phage system and protocols, to
convert a positive plaque to pBluescript.TM. plasmid DNA and
sequencing was conducted following preparation of DNA with a Qiagen
Qiaprep.TM. spin mini prep kit using the manufacturer's
instructions and restriction enzyme digestion with about 1 .mu.l of
20 U/.mu.l each of EcoRI and XhoI, available from New England
Biolabs. A clone was isolated from a secondary plaque, containing a
nucleic acid molecule of about 2240 base pairs, referred to herein
as n nCfLGIC.sub.2240, having a nucleotide sequence denoted herein
as SEQ ID NO:19. The complement of SEQ ID NO:19 is represented
herein as SEQ ID NO:21. Sequencing of nCfLGIC.sub.376 indicates
that nCfLGIC.sub.376 shared 100% identity with nucleotides 763
through 1138 of SEQ ID NO:19.
[0274] Translation of SEQ ID NO:19 suggests that nucleic acid
molecule nCfLGIC.sub.2240 encodes a partial-length LGIC protein of
569 amino acids, referred to herein as PCfLGIC.sub.569, having an
amino acid sequence represented by SEQ ID NO:20, assuming the
initiation codon spans from nucleotide 1 through nucleotide 3 of
SEQ ID NO:19 and the termination codon spans from nucleotide 1708
through nucleotide 1710 of SEQ ID NO:19. The coding region encoding
PCfLGIC.sub.569, is represented by nucleic acid molecule
nCfLGIC.sub.1707, having a coding strand with the nucleic acid
sequence represented by SEQ ID NO:22 and a complementary strand
with nucleic acid sequence represented by SEQ ID NO:24. The amino
acid sequence of PCfLGIC.sub.569, also represented as SEQ ID NO:23,
predicts that PCfLGIC.sub.569 has an estimated molecular weight of
about 64 kDa and an estimated pI of about 6.6.
[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 Rattus
norvegicus glycine receptor alpha-3 chain precursor protein,
GenBank Accession No. 121580. Comparison of SEQ ID NO:22 with
nucleic acid sequences reported in GenBank indicates that SEQ ID
NO:22 showed the most homology, i.e., about 38% identity, with a
human glycine receptor alpha-3 subunit nucleic acid molecule,
GenBank Accession number AF017715. Percent identity calculations
were performed using GCG version 9.0 using default parameters.
[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
.alpha.-.sup.32P-ATP labeled nCfLGIC.sub.376 under the Northern
Blotting conditions described in Example 4. Analysis of the
developed film revealed that LGIC mRNA was expressed in HMT tissues
but not in non-HMT tissues.
[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.degree. C. for 30
seconds, (2) 5 cycles of 94.degree. C. for 10 seconds then
72.degree. C. for 4 minutes, (3) 5 cycles of 94.degree. C. for 10
seconds then 70.degree. C. for 4 minutes, and (4) 25 cycles of
94.degree. C. for 10 seconds then 68.degree. 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.sub.613 and
having a coding sequence denoted SEQ ID NO:1859, and a
complementary strand denoted herein as SEQ ID NO:1860 was sequenced
using an ABI PRISM 377 automatic DNA Sequencer, available from
Perkin Elmer, Branchburg, N.J. Sequence analysis revealed that
nucleotides 503-613 of nCfLGIC.sub.613 had 100% identity with
nucleotides 1-110 of SEQ ID NO:19. The two sequences were aligned
to form a 2739 nucleotide contiguous sequence, referred to herein
as nCfLGIC.sub.2739, having a coding strand denoted herein as SEQ
ID NO:1861 and a complementary strand denoted herein as SEQ ID
NO:1863. Translation of SEQ ID NO:1861 suggests that nucleic acid
molecule nCfLGIC.sub.2739 encodes a full-length LGIC protein of 672
amino acids, referred to herein as PCfLGIC.sub.672, having an amino
acid sequence represented by SEQ ID NO:1862, assuming the
initiation codon spans from nucleotide 191 through nucleotide 193
of SEQ ID NO:1861 and the termination codon spans from nucleotide
2207 through nucleotide 2209 of SEQ ID NO:1861. The coding region
encoding PCfLGIC.sub.672, is represented by nucleic acid molecule
nCfLGIC.sub.2016, having a coding strand with the nucleic acid
sequence represented by SEQ ID NO:1864 and a complementary strand
with nucleic acid sequence represented by SEQ ID NO:1866. The amino
acid sequence of PCfLGIC.sub.672, i.e. SEQ ID NO:1862, predicts
that PCfLGIC.sub.672 has an estimated molecular weight of about
75.5 kDa and an estimated pI of about 5.89.
[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 Rattus norvegicus
(Accession #P24524). Comparison of SEQ ID NO:1864 with nucleic acid
sequences reported in GenBank indicates that SEQ ID NO:1864 showed
the most homology, i.e., about 43.1 % identity with the Homo
sapiens glycine receptor, alpha3 cDNA (Accession #NP006520).
Percent identity calculations were performed using GCG version 9.0
using default parameters.
[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.TM. 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.degree. C. for 30 seconds, (2) 25
cycles of 94.degree. C. for 10 seconds, 55.degree. C. for 10
seconds, and 72.degree. 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.sub.1300 was cut from the gel and purified using
the QIAquick Gel Extraction Kit as described above.
[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.degree.. 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.sub.1300 was isolated. DNA sequence analysis of
pIB/V5-His-nCfLGIC.sub.1300 confirmed that nucleotides 188-1464 of
SEQ ID NO:1861 had been successfully ligated into the pIf/V5-His
expression vector in frame with the C-terminal V5 epitope encoded
by the vector.
EXAMPLE 8
[0282] This example describes the cloning and sequencing of a C.
felis ANON/23DA nucleic acid molecule. This example also describes
the expression of ANON/23DA mRNA in a variety of flea tissues.
[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 Drosophila melanogaster. This
clone was digested with EcoRI to excise an insert about 177
nucleotides in length, referred to as flea ANON nucleic acid
molecule nCfANON.sub.177. The insert was isolated by gel
purification using a Gel Purification kit, available from Qiagen.
Approximately 50 ng of purified nCfANON.sub.177 was used to
construct a .sup.32P .alpha.-dATP labeled DNA probe using a
Megaprime DNA labeling kit, available from Amersham, using the
manufacturer's protocols.
[0284] The .sup.32P .alpha.-dATP labeled probe was used in a plaque
lift hybridization procedure to isolate a clone from the HMT
lambda-ZAP unsubtracted cDNA library described in Example 1, using
standard hybridization conditions described in Example 4. Plaques
that hybridized strongly to the probe were isolated and subjected
to in vivo excision. In vivo excision was performed using the
Stratagene Ex-Assist.TM. helper phage system and protocols, to
convert a positive plaque to pBluescript.TM. plasmid DNA and
sequencing of DNA was conducted following preparation with a Qiagen
Qiaprep.TM. spin mini prep kit using the manufacturer's
instructions and restriction enzyme digestion with about 1 .mu.l of
20 U/.mu.l each of EcoRI and XhoI, available from New England
Biolabs. A clone was isolated from a secondary plaque, containing a
nucleic acid molecule of about 1429 base pairs, referred to herein
as nCfANON.sub.1429, having a nucleotide sequence denoted herein as
SEQ ID NO:25. The complement of SEQ ID NO:25 is represented herein
as SEQ ID NO:27. Sequencing of nCfANON.sub.177 indicates that
nCfANON.sub.177 shared 100% identity with nucleotides 279 through
455 of SEQ ID NO:25.
[0285] Translation of SEQ ID NO:25 suggests that nucleic acid
molecule nCfANON.sub.1429 encodes a full-length ANON protein of 398
amino acids, referred to herein as PCfANON.sub.398, having an amino
acid sequence represented by SEQ ID NO:26, assuming the initiation
codon spans from nucleotide 18 through nucleotide 20 of SEQ ID
NO:25 and the termination codon spans from nucleotide 1212 through
nucleotide 1214 of SEQ ID NO:25. The coding region encoding
PCfANON.sub.398, is represented by nucleic acid molecule
nCfANON.sub.1194, having a coding strand with the nucleic acid
sequence represented by SEQ ID NO:28 and a complementary strand
with nucleic acid sequence represented by SEQ ID NO:30. The amino
acid sequence of PCfANON.sub.398, also represented as SEQ ID NO:29,
predicts that PCfANON.sub.398has an estimated molecular weight of
about 45 kDa and an estimated pI of about 8.8.
[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
Drosophila melanogaster ANON/23DA protein, GenBank Accession No.
924937. Comparison of SEQ ID NO:28 with nucleic acid sequences
reported in GenBank indicates that SEQ ID NO:28 showed the most
homology, i.e., about 60% identity, with a Drosophila melanogaster
ANON/23DA nucleic acid molecule, GenBank Accession number U29170.
Percent identity calculations were performed using GCG version 9.0
using default parameters.
[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
.alpha.-.sup.32P-ATP labeled nCfANON.sub.177 under the Northern
Blotting conditions described in Example 4. Analysis of the
developed film revealed that ANON mRNA was expressed in non-HMT
tissues but not in HMT tissues. ANON mRNA was also detected in all
adult fleas tested regardless of feeding conditions and in the
wandering larvae and pupal life stages.
EXAMPLE 9
[0288] This example describes the cloning and sequencing of a C.
felis malvolio nucleic acid molecule.
[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.sub.432. The insert was isolated
by gel purification using a Gel Purification kit, available from
Qiagen and sequenced using standard sequencing methods and shown to
have homology to a malvolio gene from Drosophila melanogaster,
hereinafter referred to as a flea MALV nucleic acid molecule.
[0290] Sequence information from nCfMALV.sub.432 was used to design
PCR primers to amplify a C. felis MALV nucleic acid molecule from
the HMT unsubtracted library described in Example 1 using a nested
PCR as follows. Sense primer MALV RI, having the nucleotide
sequence 5' CCA TTA TTA ACC TGG TCG ACC AC 3', designated SEQ ID
NO:41 and corresponding to nucleotides 365-387 of nCfMALV.sub.432
and reverse primer M13 Reverse, having the nucleotide sequence 5'
GGA AAC AGT ATG ACC ATG 3', designated SEQ ID NO:42 were used in a
first PCR reaction using HMT unsubtracted library as the template
using standard PCR reaction and thermocycling conditions, with the
exception that 2 .mu.l of template was used. The reaction product
from the first PCR reaction was diluted 1:50 and used as the
template in a second PCR reaction as follows. Reverse primer
malvolio R2, having a nucleotide sequence 5' CGC TAT AGT CGG TAG
GGT CGC 3', designated SEQ ID NO:43 and corresponding to
nucleotides 239-259 of nCfMALV.sub.432 and forward primer T3,
having a nucleotide sequence 5' AAT TAA CCC TCA CTA AAG GG 3' were
used in a second PCR reaction under standard PCR reaction and
thermocycling conditions.
[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.TM., Original TA cloning vector, available from
Invitrogen. The ligation reaction was then used to transform
INV.alpha.F' One Shot.TM. competent cells, available from
Invitrogen, which were plated on LB agar with 50 micrograms per
milliliter (.mu.g/ml) ampicillin, available from Sigma-Aldrich Co.,
and 50 .mu.g/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.sub.765,
having a nucleotide sequence denoted herein as SEQ ID NO:31. The
complement of SEQ ID NO:31 is represented herein as SEQ ID
NO:33.
[0292] Translation of SEQ ID NO:31 suggests that nucleic acid
molecule nCfMALV.sub.765 encodes a partial-length MALV protein of
254 amino acids, referred to herein as PCfMALV.sub.254, having an
amino acid sequence represented by SEQ ID NO:32, assuming the
initiation codon spans from nucleotide 2 through nucleotide 4 of
SEQ ID NO:31 and the last codon spans from nucleotide 761 through
nucleotide 763 of SEQ ID NO:31. The coding region encoding
PCfALV.sub.254, is represented by nucleic acid molecule
nCfMALV.sub.762, having a coding strand with the nucleic acid
sequence represented by SEQ ID NO:34 and a complementary strand
with nucleic acid sequence represented by SEQ ID NO:36. The amino
acid sequence of PCfMALV.sub.254, also represented as SEQ ID NO:35,
predicts that PCfMALV.sub.254 has an estimated molecular weight of
about 36 kDa and an estimated pI of about 4.9.
[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
Drosophila melanogaster malvolio protein, GenBank Accession No.
780776. Comparison of SEQ ID NO:34 with nucleic acid sequences
reported in GenBank indicates that SEQ ID NO:34 showed the most
homology, i.e., about 63% identity, with a Drosophila melanogaster
malvolio nucleic acid molecule, GenBank Accession number U23948.
Percent identity calculations were performed using GCG version 9.0
using default parameters.
EXAMPLE 10
[0294] This example describes the cloning, sequencing, and
recombinant expression of a C. felis odorant-binding protein-like
(OS-D) nucleic acid molecule. This example also describes the
expression of OS-D mRNA in a variety of flea tissues.
[0295] A C. felis OS-D nucleic acid molecule of about 311
nucleotides was isolated from a cat blood-fed adult flea cDNA
library, prepared as described in example 8 of PCT publication WO
96/11706 by Grieve et al., published Apr. 25, 1996, by PCR
amplification as follows. Sense primer 5'newBsaI5', having a
nucleotide sequence 5' CAA AAC TGG TCT CCC CGC TC 3', denoted SEQ
ID NO:57 was used in combination with vector primer T7, having a
nucleic acid sequence 5' TAA TAC GAC TCA CTA TAG GG 3', denoted SEQ
ID NO:58, in a first PCR reaction using the cat blood-fed adult
flea cDNA library as the template under standard PCR reaction and
thermocycling conditions. A 311-nucleotide fragment, denoted
nCfOSD.sub.311 was isolated and shown to encode a partial length
protein of 45 amino acids having a sequence similar to Drosophila
melanogaster OS-D protein. Since primer 5'newBsaI5' was designed to
be specific for the C. felis serpin constant region,
nCfOSD.sub.311, is believed to have been fortuitously amplified in
this PCR reaction.
[0296] To isolate a flea OS-D nucleic acid molecule encoding a
full-length OS-D protein, nucleic acid molecule nCfOSD.sub.311 was
used to design primers for a nested PCR as follows. Sense primer
OSD-R1, having a nucleotide sequence 5' GGT TCG CCT CTC TTC ACT TG
3', which is complementary in sequence to nucleotides 108-127 of
nCfOSD.sub.311, denoted SEQ ID NO:59, was used in combination with
M13 reverse primer, SEQ ID NO:54, in a first PCR reaction using the
cat blood-fed adult C. felis cDNA library as the template. The
product of the first reaction was diluted 1:50 and used as the
template for a second PCR reaction using reverse primer OSD-R2,
having a nucleotide sequence 5' CGG TTG GAT CGT AAA CTG CAG 3',
which is complementary in sequence to nucleotides 52-72 of
nCfOSD.sub.311, denoted SEQ ID NO:60, and forward primer T3, SEQ ID
NO:56. Each PCR reaction was conducted under standard PCR reaction
and thermocycling conditions with the exception that an annealing
temperature of 55.degree. C. was used rather than 50.degree. C.
[0297] A DNA fragment of about 365 nucleotides, referred to herein
as nCfOSD.sub.365, was isolated from the second PCR product and
purified using a Gel Purification Kit, available from Qiagen. The
purified fragment was ligated into the pCRII.TM. TA cloning vector,
available from Invitrogen, and sequenced using standard sequencing
methods. Sequencing revealed that nucleotides 294-365 of
nCfOSD.sub.365 match nucleotides 1-72 of molecule nCfOSD.sub.311,
described above. The sequences from the partial length clones
described were aligned to produce a sequence including a
full-length coding region of 604 nucleotides, referred to as
nCfOSD.sub.604, denoted herein as SEQ ID NO:37, where
nCfOSD.sub.311 is identical in sequence to nucleotides 294-604 of
SEQ ID NO:37 and nCfOSD.sub.365 is identical in sequence to
nucleotides 1-365 of SEQ ID NO:37. The complement of SEQ ID NO:37
is represented herein as SEQ ID NO:39.
[0298] Translation of SEQ ID NO:37 suggests that nucleic acid
molecule nCfOSD.sub.604 encodes a full-length OS-D protein of 135
amino acids, referred to herein as PCfOSD.sub.135, having an amino
acid sequence represented by SEQ ID NO:38, assuming the initiation
codon spans from nucleotide 26 through nucleotide 28 of SEQ ID
NO:37 and the termination codon spans from nucleotide 431 through
nucleotide 433 of SEQ ID NO:37. The coding region encoding
PCfOSD.sub.135, is represented by nucleic acid molecule
nCfOSD.sub.405, having a coding strand with the nucleic acid
sequence represented by SEQ ID NO:40 and a complementary strand
with nucleic acid sequence represented by SEQ ID NO:42. The amino
acid sequence of PCfOSD.sub.135, also represented as SEQ ID NO:41,
predicts that PCfOSD.sub.135 has an estimated molecular weight of
about 15 kDa and an estimated pI of about 8.6. Analysis of SEQ ID
NO:38 suggests the presence of a signal peptide encoded by a
stretch of amino acids spanning from about amino acid 1 through
about amino acid 20. The proposed mature protein, denoted herein as
PCfOSD.sub.115, contains about 115 amino acids corresponding to
amino acids 21 through 135 of SEQ ID NO:38. The predicted pI of the
mature protein (i.e. the protein with the signal peptide removed)
is 6.6.
[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
Schistocerca gregaria chemo sensory protein CSP-sg4, GenBank
Accession No. 3283938. Comparison of SEQ ID NO:40 with nucleic acid
sequences reported in GenBank indicates that SEQ ID NO:40 showed
the most homology, i.e., about 58% identity, with a Schistocerca
gregaria chemosensory protein CSP-sg4 nucleic acid molecule,
GenBank Accession number AF070964. Comparison of SEQ ID NO:40 with
nucleic acid molecules sequenced when screening the HNC subtracted
and unsubtracted libraries described in Example 2 revealed that
OS-D (i.e. SEQ ID NO:40) is expressed in each of these libraries.
Additional sequence analysis revealed that there are four cysteines
present in C. felis OS-D which are conserved in sequence alignments
with the four cysteines of OS-D-like molecules of other insects,
including D. melanogaser OS-D protein GenBank Accession No. U02546,
S. gregaria chemosensory protein CSP-sg4, GenBank Accession number
AF070964, and cockroach leg regenerative protein, GenBank Accession
No. AF030340. Percent identity calculations and additional sequence
analysis was performed using GCG version 9.0 using default
parameters.
[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.TM. 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.sub.357, was transformed into E. coli strain BL21,
available from Novagen, to form recombinant cell E.
coli:pTrc-nCfOSD.sub.357.
[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
C. felis having a male to female ratio of 1:4. Total RNA was
separately extracted from HNC tissues and the HNC-less carcasses
that resulted from these dissections using a standard guanidine
lysis method, described by Sambrook et al., ibid.
[0303] Approximately 15 .mu.g 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. The membrane was UV
cross-linked using the Stratalinker.RTM., available from
Stratagene, and placed in approximately 30 ml of hybridization
buffer consisting of 5.times. SSPE, 1% Sarcosyl, 50% formamide,
5.times. Denhardt's reagent and 25 mM EDTA at 42.degree. C. for
approximately 3 to 6 hours. A probe comprising the flea OS-D
nucleic acid molecule nCfOSD.sub.357 was labeled with
.alpha.-.sup.32P-ATP using a DNA labeling kit, available from
Amersham and added to the buffer at a concentration of
approximately 10.times.10.sup.6 cpm/ml, and allowed to hybridize
for about 14 to 18 hours at 42.degree. C. The blot was then washed
twice for 10 minutes per wash in 0.5.times. SSPE and 0.1% sarcosyl
at 55.degree. C. and exposed to film for autoradiography. Analysis
of the developed film showed that there was greater expression of
OS-D mRNA in HNC tissues compared to non-HNC tissues, indicating
possible upregulation of OS-D in flea head and nerve cords.
EXAMPLE 11
[0304] This example describes the cloning and sequencing of a C.
felis N-methyl-D-aspartate receptor associated (NMDA) nucleic acid
molecule.
[0305] 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 NMDA genes. This clone was digested with
EcoRI to excise an insert 279 nucleotides in length, referred to as
flea NMDA nucleic acid molecule nCfNMDA.sub.279. The insert was
isolated by gel purification using a Gel Purification kit,
available from Qiagen. Approximately 50 ng of purified
nCfNMDA.sub.279 was used to construct a .sup.32P .alpha.-dATP
labeled DNA probe using a Megaprime DNA labeling kit, available
from Amersham, using the manufacturer's protocols.
[0306] The .sup.32P .alpha.-dATP labeled probe was used in a plaque
lift hybridization procedure to isolate a clone from the HMT
lambda-ZAP unsubtracted cDNA library described in Example 1, using
standard hybridization conditions described in Example 4. Plaques
that hybridized strongly to the probe were isolated and subjected
to in vivo excision. In vivo excision was performed using the
Stratagene Ex-Assist.TM. helper phage system and protocols, to
convert a positive plaque to pBluescript.TM. plasmid DNA and
sequencing was conducted following preparation of DNA with a Qiagen
Qiaprep.TM. spin mini prep kit using the manufacturer's
instructions and restriction enzyme digestion with about 1 .mu.l of
20 U/.mu.l each of EcoRI and XhoI, available from New England
Biolabs. A clone was isolated from a secondary plaque, containing a
nucleic acid molecule of about 1227 base pairs, referred to herein
as nCfNMDA.sub.1227, having a nucleotide sequence denoted herein as
SEQ ID NO:43. The complement of SEQ ID NO:43 is represented herein
as SEQ ID NO:45. Sequencing of nCfNMDA.sub.279 indicates that
nCfMDA.sub.279 shared 100% identity with nucleotides 709 through
987 of SEQ ID NO:43.
[0307] Translation of SEQ ID NO:43 suggests that nucleic acid
molecule nCfNMDA.sub.1227 encodes a full-length NMDA protein of 246
amino acids, referred to herein as PCfNMDA.sub.246, having an amino
acid sequence represented by SEQ ID NO:44, assuming the initiation
codon spans from nucleotide 312 through nucleotide 314 of SEQ ID
NO:43 and the termination codon spans from nucleotide 1050 through
nucleotide 1052 of SEQ ID NO:43. The coding region encoding
PCfNMDA.sub.246, is represented by nucleic acid molecule
nCfNMDA.sub.738, having a coding strand with the nucleic acid
sequence represented by SEQ ID NO:46 and a complementary strand
with nucleic acid sequence represented by SEQ ID NO:48. The amino
acid sequence of PCfNMDA.sub.246, also represented as SEQ ID NO:47
predicts that PCfNMDA.sub.246has an estimated molecular weight of
about 27 kDa and an estimated pI of about 5.6.
[0308] Comparison of amino acid sequence SEQ ID NO:44 with amino
acid sequences reported in GenBank indicates that SEQ ID NO:44
showed the most homology, i.e., about 34% identity, with a
Emericella nidulans negative-acting regulatory protein, GenBank
Accession No. 3676056. Comparison of SEQ ID NO:46 with nucleic acid
sequences reported in GenBank indicates that SEQ ID NO:46 showed
the most homology, i.e., about 45% identity, with a Drosophila
melanogaster NMDA nucleic acid molecule, GenBank Accession number
L37377. Percent identity calculations were performed using GCG
version 9.0 using default parameters.
EXAMPLE 12
[0309] This example describes the cloning and sequencing of C.
felis chemical sense related lipophilic ligand binding protein
nucleic acid molecule. This example also describes the expression
of chemical sense related lipophilic ligand binding protein mRNA in
a variety of flea tissues.
[0310] A TA clone from the HNC EST library described in Example 2
was sequenced using standard sequencing methods and shown to have
significant homology to chemical sense related lipophilic ligand
binding protein (CLBP) genes. This clone was digested with EcoRI to
excise an insert 339 nucleotides in length, referred to as flea
CLBP nucleic acid molecule nCfCLBP.sub.339. The insert was isolated
by gel purification using a Gel Purification kit, available from
Qiagen, Chatsworth, CA. Approximately 50 ng of purified
nCfCLBP.sub.339 was used to construct a .sup.32P .alpha.-dATP
labeled DNA probe using a Megaprime DNA labeling kit, available
from Amersham, using the manufacturer's protocols.
[0311] The .sup.32P .alpha.-dATP labeled probe was used in a
standard plaque lift hybridization procedure to isolate a clone
from the HNC lambda-ZAP unsubtracted cDNA library described in
Example 2. The following hybridization conditions were used.
Filters were hybridized with about 5.times.10.sup.7 counts per
minute (cpm) per ml of the probe in 100 ml of buffer (5.times.
SSPE, 1 % Sarcosyl, 0.1 mg/ml BLOTTO) at 45.degree. C. for about 14
hours. The filters were washed twice for 20 minutes per wash in 500
ml of 0.5.times. SSPE and 0.1% Sarcosyl at 55.degree. C. and
subjected to autoradiography. Two plaques that hybridized strongly
to the probe were isolated and subjected to in vivo excision using
the Stratagene Ex-Assist.TM. helper phage system and protocols.
Miniprep DNA was prepared from each positive clone using a Quantum
Prep mini prep kit, available from BioRad, Hercules, Calif., and
sequenced using standard sequencing procedures. Sequencing revealed
that the two positive clones share 97% amino acid identity to each
other. The first clone contained a nucleic acid molecule of about
633 nucleotides, referred to herein as nCfCLBP1A.sub.633, having a
nucleotide sequence denoted herein as SEQ ID NO:153. The complement
of SEQ ID NO:153 is represented herein as SEQ ID NO:155. The second
clone contained a nucleic acid molecule of about 631 nucleotides,
referred to herein as nCfCLBP2A.sub.631, having a nucleotide
sequence denoted herein as SEQ ID NO:162. The complement of SEQ ID
NO:162 is represented herein as SEQ ID NO:164. Sequencing of
nCfCLBP.sub.340 indicated that nCfCLBP.sub.339 shared 100% identity
with nucleotides 1 through 339 of SEQ ID NO:153 and shared 100%
identity with nucleotides 2 through 339 of SEQ ID NO:162.
[0312] Translation of SEQ ID NO:153 suggests that nucleic acid
molecule nCfCLBP1A.sub.633 encodes a full-length CLBP protein of
147 amino acids, referred to herein as PCfCLBP1A.sub.147, having an
amino acid sequence represented by SEQ ID NO:154, assuming the
initiation codon spans from nucleotide 67 through nucleotide 69 of
SEQ ID NO:153 and the termination codon spans from nucleotide 511
through nucleotide 513 of SEQ ID NO:153. The coding region encoding
PCfCLBP1A.sub.147, is represented by nucleic acid molecule
nCfCLBP1A.sub.441, having a coding strand with the nucleic acid
sequence represented by SEQ ID NO:156 and a complementary strand
with nucleic acid sequence represented by SEQ ID NO:158. The amino
acid sequence of PCfCLBP1A.sub.147, also represented by SEQ ID
NO:157, predicts that PCfCLBP1A.sub.147 has an estimated molecular
weight of about 15 kDa and an estimated pI of about 5.
[0313] Analysis of SEQ ID NO:154 suggests the presence of a signal
peptide encoded by a stretch of amino acids spanning from about
amino acid 1 through about amino acid 19. The proposed mature
protein, denoted herein as PCfCLBP1A.sub.12, contains 128 amino
acids which is represented herein as SEQ ID NO:160.
PCfCLBP1A.sub.128 is encoded by a nucleic acid molecule denoted
nCfCLBP1A.sub.384 having a coding strand with nucleic acid sequence
SEQ ID NO:159 and a complementary strand with SEQ ID NO:161.
[0314] Translation of SEQ ID NO:162 suggests that nucleic acid
molecule nCfCLBP2A.sub.631 encodes a full-length CLBP protein of
147 amino acids, referred to herein as PCfCLBP2A.sub.147, having an
amino acid sequence represented by SEQ ID NO:163, assuming the
initiation codon spans from nucleotide 65 through nucleotide 67 of
SEQ ID NO:162 and the termination codon spans from nucleotide 509
through nucleotide 511 of SEQ ID NO:162. The coding region encoding
PCfCLBP2A.sub.147, is represented by nucleic acid molecule
nCfCLBP2A.sub.441, having a coding strand with the nucleic acid
sequence represented by SEQ ID NO:165 and a complementary strand
with nucleic acid sequence represented by SEQ ID NO:167. The amino
acid sequence of PCfCLBP2A.sub.147 predicts that PCfCLBP2A.sub.147
has an estimated molecular weight of about 15 kDa and an estimated
pI of about 5.
[0315] Analysis of SEQ ID NO:163 suggests the presence of a signal
peptide encoded by a stretch of amino acids spanning from about
amino acid 1 through about amino acid 19. The proposed mature
protein, denoted herein as PCfCLBP2A.sub.128, contains about 128
amino acids which is represented herein as SEQ ID NO:169.
PCfCLBP2A.sub.128 is encoded by a nucleic acid molecule denoted
nCfCLBP2A.sub.384 having a coding strand with nucleic acid sequence
SEQ ID NO:168 and a complementary strand with SEQ ID NO:170.
[0316] Comparison of amino acid sequences SEQ ID NO:154 and SEQ ID
NO:163 with amino acid sequences reported in GenBank indicates that
each sequence showed the most homology, i.e., about 29% identity,
with a Drosophila melanogaster pheromone binding protein related
protein 2 (PBPRP-2), GenBank Accession No. 1709595. Percent
identity calculations were performed using GCG version 9.0 using
default parameters. Blast comparison of nucleic acid sequences SEQ
ID NO:156 and SEQ ID NO:165 with nucleic acid sequences reported in
GenBank indicates that each sequence showed the most homology to a
human Xp22 PAC PRCI1-5G11 nucleic acid molecule, GenBank Accession
number AC002369. Pairwise identity could not be performed as the
human clone in GenBank is too large to load into GCG version 9.0.
Blast comparison performed using default parameters showed an
insignificant level of identity of 0.87. Additional sequence
analysis revealed that there are six cysteines present in C. felis
CLBP which are conserved in sequence alignments with the six
cysteines of neuronal/sense-related molecules in the PBP/GOBP
family, including D. melanogaser PBPRP-2, GenBank Accession No.
1709595, and PBPRP-5, GenBank Accession No. P54195, proteins, and
Phormia regina chemical sense related lipophilic ligand binding
protein (CSRLLBP), GenBank Accession No. S65458.
[0317] A Northern Blot analysis was conducted to determine whether
CLBP mRNA is expressed exclusively in HNC tissues. HNC tissues were
dissected, total RNA was isolated and separated by electrophoresis
as described in Example 10.
[0318] Following electrophoresis, RNA was blotted as described in
Example 10 and a probe comprising clone nCfCLBP.sub.340 labeled
with .alpha.-.sup.32P-ATP was added to the buffer at a
concentration of approximately 1.times.10.sup.6 cpm/ml and allowed
to hybridize for about 14 to 18 hours. The blot was then washed as
described in Example 10 and exposed to film for autoradiography.
Analysis of the developed film showed that there was greater
expression of CLBP mRNA in HNC tissues compared to non-HNC tissues,
indicating possible upregulation of CLBP in flea head and nerve
cords.
[0319] The coding region of nCfCLBP2A.sub.631, i.e. SEQ ID NO:162,
was PCR amplified from the pBluescript.TM. clone described above as
the template, using sense primer 2A1BamSen having nucleotide
sequence 5' ATG GAT CCG GCA AAA TAT ACC AAA GAA GAA G 3', having a
BamHI site indicated in bold, designated herein as SEQ ID NO:1952,
and anti-sense primer 2A1antiR1, having nucleotide sequence 5' ATG
AAT TCT TAT ATT GGT ATC GCG TCC ATT 3', having a EcoRI site
indicated in bold, designated herein as SEQ ID NO:1953. PCR
reactions were performed using the following thermocycling
conditions: (a) one cycle at 95.degree. C. for one minute; (b) five
cycles at 94.degree. C. for ten seconds, 49.degree. C. for
twenty-five seconds, and 69.degree. C. for one minute; and (c)
twenty-three cycles at 94.degree. C. for ten seconds, 53.degree. C.
for twenty seconds, and 69.degree. C. for seventy-five seconds, in
reactions containing 0.2 mM dNTPs, 1 .mu.M of each primer, 0.5
.mu.l of 5U/.mu.l KlenTaq Advantage polymerase, available from
Clontech, 1 .mu.l of 1 .mu.g/.mu.l template, and 1.times. KlenTaq
buffer, hereinafter referred to as "standard PCR conditions". The
PCR product was digested with BamHI and EcoRI and ligated into the
vector pTrcHisB, available from Invitrogen, that had been digested
with BamHI and EcoRI. The resulting recombinant molecule, referred
to herein as pTrc-nCfCLBP2A.sub.441, was transformed into E. coli
strain BL21, available from Novagen Inc., Madison, Wis., to form
recombinant cell E. coli:pTrc-nCfCLBP2A.sub.441.
[0320] The recombinant cell was grown under standard conditions and
then incubated in the presence of 0.5
.mu.isopropylthio-.beta.-galactoside (IPTG) to induce expression of
recombinant protein. 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 18 kDa protein. The protein product was purified as
follows. The recombinant cells were collected by centrifugation,
the supernatant was discarded and the pellets were resuspended and
homogenized in 60 ml (total) of 50 mM Tris pH8.0 containing 50 mM
NaCl and 1 mM phenylmethylsulfonyl fluoride(PMSF). The sample was
then passed through the microfluidizer five times, rocked at
4.degree. C. for 20 minutes, and centrifuged at 20,000.times. G for
30 minutes. The supernatant was collected and filtered through a
0.45 um filter then run over a HiTrap Chelating column, available
from Amersham Pharmacia, in 50 mM Tris pH8 containing 50 mM NaCl
and 10 mM immidazole and eluted with an increasing imidazole
gradient. The recombinant protein was eluted at approximately 150
mM imidazole. Fractions containing recombinant protein were pooled
and concentrated using a Centricon Plus-20 (Amicon), and
diafiltered into PBS. Quantification of the protein was performed
by densitometry against a known standard.
EXAMPLE 13
[0321] This Example describes the further characterization and
expression of a Sodium/Hydrogen Transporter-like cDNA isolated by
EST sequencing described in Example 1.
[0322] A cDNA designated clone 2231-94 was isolated from the
unsubtracted HMT library as described in Example 1. Analysis of
clone 2231-94 indicated that the cDNA, denoted nCfNAH.sub.2080, is
about 2080 nucleotides in length, having a coding strand with
nucleic acid sequence SEQ ID NO:1867 and a complementary sequence
having SEQ ID NO:1869. Translation of SEQ ID NO:1867 suggests that
nucleic acid molecule nCfNAH.sub.2080 encodes a full-length
Sodium/Hydrogen Transporter-like protein of 608 amino acids,
referred to herein as PCfNAH.sub.608, having an amino acid sequence
represented by SEQ ID NO:1868, assuming the initiation codon spans
from nucleotide 45 through nucleotide 47 of SEQ ID NO:1867 and the
termination codon spans from nucleotide 1869 through nucleotide
1871 of SEQ ID NO:1867. The coding region encoding PCfNAH.sub.608,
is represented by nucleic acid molecule nCfNAH.sub.1824, having a
coding strand with the nucleic acid sequence represented by SEQ ID
NO:1870 and a complementary strand with nucleic acid sequence
represented by SEQ ID NO:1871. The amino acid sequence of SEQ ID
NO:1868, predicts that PCfNAH.sub.608 has an estimated molecular
weight of about 67.9 kDa and an estimated isoelectric point (pI) of
about 6.47.
[0323] Comparison of amino acid sequence SEQ ID NO:1868 with amino
acid sequences reported in GenBank indicates that SEQ ID NO:1868
showed the most homology, i.e., about 67.7% identity, with a sodium
hydrogen exchanger NHE1 (Accession #AAD32689.1). Comparison of SEQ
ID NO:1867 with nucleic acid sequences reported in GenBank
indicates that SEQ ID NO:1867 showed the most homology, i.e., about
59.5% identity, with a Drosophila melanogaster sodium hydrogen
exchanger NHE1 (Accession #AF142676). Percent identity calculations
were performed using GCG version 9.0 using default parameters.
[0324] In order to express the full-length putative NaH protein,
the entire coding region was amplified by PCR and then ligated into
the InsectSelect.TM. expression vector pIB/V5-His, available from
Invitrogen, as follows. Forward primer NaH-IS-FE, which corresponds
to nucleotides 42-74 of SEQ ID NO:1867, having the sequence 5' GAC
TAG TAA AAT GGG CGT TAA AAA TAT ATA TTT ATA CTG C 3', denoted SEQ
ID NO:1939 and having a SpeI site indicated in bold, was used in
conjunction with reverse primer NaH-IS-RE, which is complementary
to nucleotides 1845-1867 of SEQ ID NO:1867, having the sequence 5'
CCG CTC GAG GTA CTG CAC GTA CTA ACG TCA TC 3', denoted SEQ ID
NO:1940 and having a XhoI restriction site indicated in bold, in a
PCR reaction using SEQ ID NO:1867 as the template. Standard PCR
reaction conditions were used with the following thermocycling
conditions: (1) 94.degree. C. for 30 seconds, (2) 25 cycles of
94.degree. C. for 10 seconds, 55.degree. C. for 10 seconds and
72.degree. C. for 3 minutes. The products of this reaction were
separated on a 1.5% agarose gel, and a band corresponding to an
approximately 1825 nucleotide molecule was cut from the gel and
purified using the QIAquick Gel Extraction Kit as described above.
The PCR product was then digested with SpeI and XhoI restriction
endonucleases for 18 hours at 37.degree.. 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 SpeI and XhoI and treated with shrimp alkaline
phosphatase, available from New England BioLabs, Inc., for 30
minutes at 37.degree.. Following standard transformation
procedures, a bacterial clone containing the plasmid pIB/V5-His-NaH
was isolated. DNA sequence analysis of the clone confirmed that
nucleotides 42-1867 of SEQ ID NO:1867, referred to herein as
nCfNAH.sub.1826, had been successfully ligated into the pIB/V5-His
expression vector in frame with the C-terminal V5 epitope encoded
by the vector.
[0325] A Northern Blot analysis was conducted as described in
Example 4 to determine whether NaH mRNA is expressed only in
certain life stages of the flea life cycle and whether NaH mRNA is
expressed only in HMT tissue. Total RNA was extracted from eggs,
first, third, and wandering larvae, pupae, unfed adults, and adults
fed on cat blood for 0.25, 2, 8, and 24 hours. In addition, total
RNA was extracted from hindguts and Malpighian tubules extracted
from 24 hour cat blood-fed adult fleas, and from the remaining body
parts following the removal of hindguts and Malpighian tubules.
Each RNA sample was separated by gel electrophoresis, transferred
to nylon membranes and hybridized with .alpha.-.sup.32P-ATP labeled
nCfNAH.sub.1826 under the Northern Blotting conditions described in
Example 4. Analysis of the developed film revealed that NAH mRNA
was expressed in the 0.25, 2, and 8 hour adult fed timepoints
only.
EXAMPLE 14
[0326] This Example describes the further characterization of a
Chloride Intracellular Channel-like cDNA isolated by EST sequencing
described in Example 1.
[0327] A cDNA designated clone 2233-24 was isolated from the
unsubtracted HMT library as described in Example 1. Analysis of
clone 2233-24 indicated that the cDNA, denoted nCfCLIC.sub.2283 is
about 2283 nucleotides in length, having a coding strand with
nucleic acid sequence SEQ ID NO:1872 and a complementary sequence
having SEQ ID NO:1874. Translation of SEQ ID NO:1872 suggests that
nucleic acid molecule nCfCLIC.sub.2283 encodes a full-length
Chloride Intracellular Channel-like protein of 262 amino acids,
referred to herein as PCfCLIC.sub.262, having an amino acid
sequence represented by SEQ ID NO:1873, assuming the initiation
codon spans from nucleotide 60 through nucleotide 62 of SEQ ID
NO:1872 and the termination codon spans from nucleotide 846 through
nucleotide 848 of SEQ ID NO:1872. The coding region encoding
PCfCLIC.sub.262, is represented by nucleic acid molecule
nCfCLIC.sub.786, having a coding strand with the nucleic acid
sequence represented by SEQ ID NO:1875 and a complementary strand
with nucleic acid sequence represented by SEQ ID NO:1876. The amino
acid sequence of SEQ ID NO:1873, predicts that PCfCLIC.sub.262 has
an estimated molecular weight of about 30.2 kDa and an estimated
isoelectric point (pI) of about 6.02.
[0328] Comparison of amino acid sequence SEQ ID NO:1873 with amino
acid sequences reported in GenBank indicates that SEQ ID NO:1873
showed the most homology, i.e., about 37.8% identity, with a Homo
sapiens chloride intracellular channel 2 (Accession #NP001280.1).
Comparison of SEQ ID NO:1872 with nucleic acid sequences reported
in GenBank indicates that SEQ ID NO:1872 showed the most homology,
i.e., about 37.5% identity with a Homo sapiens chloride
intracellular channel 2 (Accession #NM001289). Percent identity
calculations were performed using GCG version 9.0 using default
parameters.
EXAMPLE 15
[0329] This Example describes the further characterization of a
Peritrophin-like cDNA, referred to herein as PL2, isolated by EST
sequencing described in Example 1.
[0330] A cDNA designated clone 2232-23 was isolated from the
unsubtracted HMT library as described in Example 1, denoted herein
as SEQ ID NO:1877. Analysis of clone 2232-23 indicated that the
cDNA, denoted nCfPL2.sub.457 is about 457 nucleotides in length.
Translation of the coding strand of nCfPL2.sub.457 suggests that
nucleic acid molecule nCfPL2.sub.457 encodes a partial-length
Peritrophin-like protein of 113 amino acids, referred to herein as
PCfPL2.sub.113, assuming a stop coding at nucleotides 342-344 of
nCfPL2.sub.457.
[0331] Additional coding sequence corresponding to the 5' end of
nCfPL2.sub.457 was isolated by PCR performed using a RACE cDNA pool
prepared as described in Example 3 as template. A first PCR
reaction was performed using reverse primer PL2-R1, which is
complementary to nucleotides 167-187 of the nCfPL2.sub.457 cDNA,
having a nucleic acid sequence 5' GTC TGG AAG CTC AGG AAG AGG 3',
denoted herein as SEQ ID NO:1941, in conjunction with forward
Adapter Primer 1, SEQ ID NO:1933, described above under the
following thermocycling conditions: (1) 94.degree. C. for 30
seconds, (2) 5 cycles of 94.degree. C. for 10 seconds and
72.degree. C. for 4 minutes, (3) 5 cycles of 94.degree. C. for 10
seconds and 70.degree. C. for 4 minutes, and (4) 25 cycles of
94.degree. C. for 10 seconds then 68.degree. C. for 4 minutes. The
product of this reaction was diluted 1:50 and used as template for
a second PCR reaction as follows. Forward adapter primer 2, SEQ ID
NO:1935, was used with reverse primer PL2-R2, which is
complementary to nucleotides 29-52 of the nCfPL2.sub.457 cDNA,
having a nucleic acid sequence 5' GTA ATA TGC GTG ACA ATC GTG TGG
3', denoted herein as SEQ ID NO:1942, using the thermocycling
conditions described for the first PCR reaction. The resulting
product was gel purified as described above to reveal a distinct
band corresponding to nucleic acid molecule of approximately 900 bp
in length. The fragment was then ligated into the pCR II TA Cloning
vector, available from Qiagen and sequenced using an ABI PRISM 377
automatic DNA Sequencer. Sequencing revealed that nucleotides
791-835 of the fragment had 100% identity with nucleotides 1-45 of
the nCfPL2.sub.457 cDNA. The two sequences were aligned to form a
contiguous sequence, denoted nCfPL2.sub.1291, which is about 1291
nucleotides in length, having a coding strand with nucleic acid
sequence SEQ ID NO:1878 and a complementary sequence having SEQ ID
NO:1879. Translation of SEQ ID NO:1878 suggests that nucleic acid
molecule nCfPL2.sub.1291, encodes a non full-length
Peritrophin-like protein of 391 amino acids.
[0332] In order to isolate the additional sequence corresponding to
the 5' end of SEQ ID NO:1878, nested PCR reactions were performed
using the RACE cDNA pool as template. For the first PCR, forward
adapter primer AP1 was used with reverse primer PL2-R1 under
standard PCR reaction conditions and the following thermocycling
conditions: (1) 94.degree. C. for 1 minute, (2) 5 cycles of
94.degree. C. for 20 seconds and 70.degree. C. for 1 minute, (3) 5
cycles of 94.degree. C. for 20 seconds and 68.degree. C. for 1
minute, (4) 10 cycles of 94.degree. C. for 20 seconds and
66.degree. C. for 1 minute. The products of this reaction were
diluted 1:50 in water and used as template for the second, nested
PCR. The second PCR reaction used forward adapter primer AP2 in
conjunction with reverse primer PL2-R5, which is complementary to
nucleotides 70-93 of SEQ ID NO:1878, having a nucleotide sequence
5' CGG TGC AAG TTA TAG AAC CTT CCG 3', denoted herein as SEQ ID
NO:1943 under standard PCR reaction conditions using the following
thermocycling conditions: (1) 94.degree. C. for 1 minute, (2) 5
cycles of 94.degree. C. for 20 seconds and 70.degree. C. for 1
minute, (3) 5 cycles of 94.degree. C. for 20 seconds and 68.degree.
C. for 1 minute, (4) 40 cycles of 94.degree. C. for 20 seconds and
66.degree. C. for 1 minute. The products of this reaction were
separated by agarose gel electrophoresis and a band approximately
279 nucleotides in length was excised from the gel and purified as
described above. The fragment, referred to as nCfPL2.sub.279,
having a coding nucleic acid sequence designated SEQ ID NO:1880 and
a complementary sequence designated SEQ ID NO:1881, was then
ligated into the pCROII TA Cloning vector, available from Qiagen,
and sequenced as described above. Sequencing revealed that
nucleotides 228-279 of nCfPL2.sub.279 were identical to nucleotides
42-93 of SEQ ID NO:1878, however, nucleotides 186-228 of
nCfPL2.sub.279 had no significant similarity to SEQ ID NO:1878.
This discrepancy may be the result of alternative RNA splicing or
may be an artifact of the cDNA pool. To determine the reason for
this discrepancy, additional fragments corresponding to this region
were isolated by PCR from flea cDNA libraries from adult midguts,
hindgut and Malpighian tubules and mixed instar larvae using
techniques described herein. Sequence analysis of fragments
obtained from these libraries revealed that these fragments were
identical in sequence to the sequence of nCfPL2.sub.279, therefore,
the region of SEQ ID NO:1878 which did not align to nCfPL2.sub.279
was deemed to be an artifact and was not used in subsequent
alignments.
[0333] The PL2 sequences described above were aligned to form a
contiguous sequence, denoted nCfPL2.sub.1477, which is about 1477
nucleotides in length, having a coding strand with nucleic acid
sequence SEQ ID NO:1882 and a complementary sequence having SEQ ID
NO:1884. Translation of SEQ ID NO:1882 suggests that nucleic acid
molecule nCfPL2.sub.1477 encodes a full-length Peritrophin-like
protein of 453 amino acids, referred to herein as PCfPL2.sub.453,
having an amino acid sequence represented by SEQ ID NO:1883,
assuming an initiation codon spanning from nucleotide 3 through
nucleotide 5 of SEQ ID NO:1882 and a termination codon spanning
from nucleotide 1362 through nucleotide 1364 of SEQ ID NO:1882. The
coding region encoding PCfPL2.sub.453, is represented by nucleic
acid molecule nCfPL2.sub.1359, having a coding strand with the
nucleic acid sequence represented by SEQ ID NO:1885 and a
complementary strand with nucleic acid sequence represented by SEQ
ID NO:1886. The amino acid sequence of SEQ ID NO:1883, predicts
that PCfPL2.sub.453 has an estimated molecular weight of about 49
kDa and an estimated isoelectric point (pI) of about 4.7.
[0334] Comparison of amino acid sequence SEQ ID NO:1883 with amino
acid sequences reported in GenBank indicates that SEQ ID NO:1883
showed the most homology, i.e., about 28% identity, with a
Drosophila melanogaster locus AE003474 protein (Accession #
AAF47629). Comparison of SEQ ID NO:1882 with nucleic acid sequences
reported in GenBank indicates that SEQ ID NO:1882 showed the most
homology, i.e., about 50% identity, Penaeus semisulcatus (a
crustacean) peritrophin-like protein 1 cDNA (Accession # AF095580).
Percent identity calculations were performed using GCG version 9.0
using default parameters.
EXAMPLE 16
[0335] This Example describes the further characterization and
expression of a Peritrophin-like sequence cDNA, referred to herein
as PL3, isolated by EST sequencing described in Example 1.
[0336] A cDNA designated clone 2240-17 was isolated from the
unsubtracted HMT library as described in Example 1. Analysis of
clone 2240-17 indicated that the cDNA, denoted nCfPL3.sub.406, is
about 406 nucleotides in length, having a coding strand with
nucleic acid sequence SEQ ID NO:1887 and a complementary sequence
having SEQ ID NO:1889. Translation of SEQ ID NO:1887 suggests that
nucleic acid molecule nCfPL3.sub.406 encodes a full-length
Peritrophin-like protein of 81 amino acids, referred to herein as
PCfPL81, having an amino acid sequence represented by SEQ ID
NO:1888, assuming the initiation codon spans from nucleotide 20
through nucleotide 22 of SEQ ID NO:1887 and the termination codon
spans from nucleotide 263 through nucleotide 265 of SEQ ID NO:1887.
The coding region encoding PCfPL3.sub.81, is represented by nucleic
acid molecule nCfPL3.sub.243, having a coding strand with the
nucleic acid sequence represented by SEQ ID NO:1890 and a
complementary strand with nucleic acid sequence represented by SEQ
ID NO:1891. The amino acid sequence of SEQ ID NO:1888, predicts
that PCfPL3.sub.81 has an estimated molecular weight of about 9.1
kDa and an estimated isoelectric point (pI) of about 3.64.
[0337] Comparison of amino acid sequence SEQ ID NO:1888 with amino
acid sequences reported in GenBank indicates that SEQ ID NO:1888
showed the most homology, i.e., about 34.2% identity, with a
Anopheles gambiae peritrophin 1 protein (Accession #AAC39127).
Comparison of SEQ ID NO:1887 with nucleic acid sequences reported
in GenBank indicates that SEQ ID NO :1887 showed the most homology,
i.e., about 37% identity, with a Anopheles gambiae chloride
intracellular channel 2 (Accession #AF030431). Percent identity
calculations were performed using GCG version 9.0 using default
parameters.
[0338] In order to express the full-length putative PL3 protein,
the entire coding region was amplified by PCR and then ligated into
the E. coli expression vector pTrcHisB, available from Invitrogen,
as follows. Forward primer PL3FE, which corresponds to nucleotides
70-93 of SEQ ID NO:1887, having the sequence 5' CGG GAT CCC GAA TAT
GCT GAC GTA GAT GTG TG 3', denoted SEQ ID NO:1944, and having a
BamHI restriction endonuclease site indicated in bold, was used in
conjunction with reverse primer PL3RE, which is complementary to
nucleotides 245-269 of SEQ ID NO:1887, having the sequence 5' GGA
ATT CTG TTT TAT TCT GGT TGG TAA CAT TC 3', denoted herein as SEQ ID
NO:1945 and having an EcoRI restriction endonuclease site indicated
in bold, in a PCR reaction using SEQ ID NO:1887 as the template
under standard PCR reaction conditions and the following
thermocycling conditions: (1) 94.degree. C. for 30 seconds, (2) 25
cycles of 94.degree. C. for 10 seconds, 55.degree. C. for 10
seconds and 72.degree. C. for 3 minutes. The reaction product was
separated on a 1.5% agarose gel, and a band corresponding to an
approximately 200 nucleotide molecule, as visualized by agarose gel
electrophoresis and ethidium bromide staining, was cut from the gel
and purified using the QIAquick Gel Extraction Kit as described
above.
[0339] The product of the PCR reaction was the digested with BamHI
and EcoRI restriction endonucleases, available from New England
BioLabs, Inc. for 18 hours at 37.degree. C., purified using the
QIAquick Nucleotide Removal Kit, available from Qiagen, and ligated
into the vector pTrcHisB which had been similarly digested, treated
with shrimp alkaline phosphatase, available from New England
BioLabs, Inc., for 30 minutes at 37.degree. C., and purified.
Following standard transformation procedures into E. coli BL-21
competent cells, a bacterial clone containing the plasmid
pTrcHisB-PL3 was isolated. DNA sequence analysis of the clone
confirmed that 70-269 of SEQ ID NO:1887 had been successfully
ligated into the pTrcHisB expression vector in frame with the
N-terminal T7 Tag epitope encoded by the vector. The recombinant
protein encoded thereby is predicted to be 97 amino acids in length
and have a molecular mass of 10.9 kDa, including the T7 Tag and
have a pI of 4.08.
[0340] A recombinant PL3 protein was expressed as follows. Five mls
of Luria broth were innoculated with a glycerol stock of E. coli
BL-21 competent cells, available from Novagen, Madison, Wis., that
had been transformed with the pTrcHisB-PL3 plasmid prepared as
described above and allowed to grow overnight at 37.degree. C.
under selection with 100 .mu.g/ml ampicillin. A 1 ml aliquot of
this culture was then used to inoculate 10 mls of fresh Luria broth
containing 100 .mu.g/ml ampicillin and the culture was allowed to
grow to an approximate OD reading of 0.5. A 1 ml aliquot of the
culture was removed, the cells were pelleted by centrifugation and
the supernatant discarded. The cells were resuspended in a solution
of 100 .mu.l PBS and 100 .mu.l of 2.times. SDS-PAGE loading buffer
(100 mM Tris pH 6.8, 4% SDS, 20% glycerol, 0.02% bromophenol blue,
and 10% 2-mercaptoethanol). Following removal of the 1 ml aliquot
described above, IPTG was added to the remaining 9 ml culture to a
final concentration of 5 mM of IPTG, the culture was incubated at
37.degree. C. for an additional 60 minutes, 1 ml was removed and
the OD measured at approximately 0.6. The cells in this 1 ml sample
were then pelleted by centrifugation and resuspended in a solution
of 120 .mu.l of PBS and 120 .mu.l of SDS-PAGE loading buffer. Equal
volumes of the IPTG-induced and uninduced lysates were loaded onto
a 14% Tris-Glycine SDS-PAGE gel, available from Novex, San Diego,
Calif.
[0341] Following electrophoresis, the proteins were transferred
from the SDS-PAGE gel to a nitrocellulose membrane and a Western
blot analysis was performed using the T7 tag antibody, available
from Novagen, which revealed an approximately 18 kDa protein was
induced by IPTG. The fact that the recombinant PL3 protein ran at a
higher molecular weight than predicted is consistent with previous
published results for other peritrophin proteins, and is thought to
be due in part to the characteristically low pI of these proteins
(Tellam et al., (1999) Peritrophic Matrix Proteins, Insect
Biochemistry and Molecular Biology, 29:87-101). Sequence analysis
of this protein indicates that it contained the N-terminal T7 Tag
encoded by the vector.
[0342] Four flasks, each containing 1 liter of Luria broth with 100
.mu.g/ml ampicillin were inoculated with a starter culture of 5 ml
of E. coli BL-21 cells transformed with the pTrcHisB-PL3 plasmid as
described above. The cultures were allowed to grow at 37.degree. C.
until the optical density reached approximately 0.500, at which
time a 1 ml aliquot was removed from each flask as the
pre-induction sample. IPTG was added to each 1 liter flask to a
final concentration of 0.5 mM and the cultures allowed to grow at
37.degree. C. for 135 additional minutes, at which time a 1 ml
aliquot was removed from each flask as the post-induction sample.
The 1 ml aliquots were centrifuged, the supernatants were discarded
and the pellets were resuspended in 100 .mu.l 2.times. SDS-PAGE
loading buffer per each 0.5 optical density units measured. The
pre-induction and post induction samples were then tested for
recombinant PL3 protein expression using the standard Western blot
techniques and the T7 Tag antibody described above. A protein
running at approximately 18 kDa was detected in the post-induced
but not in the pre-induced samples.
[0343] The cells from the remaining 4 liters of culture were
centrifuged, the supernatants were discarded and the cell pellets
were combined and resuspended in 120 mls of buffer A (50 mM Tris,
PH 8.0, 20 mM NaCl, 1 mM phenylmethylsulfonyl fluoride (PMSF)). The
sample was then passed through a microfluidizer five times then
rocked at 4.degree. C. for 20 minutes. The sample was then
centrifuged for 30 minutes and the supematant collected. Western
blot analysis of the supernatant showed that the recombinant PL3
protein was soluble in the first buffer A extraction. The buffer A
supernatant containing the recombinant PL3 protein was then further
purified by a nickel column, a Q2 anion exchange chromatography
column, and cation exchange chromatography, using techniques well
known to those of skill in the art.
EXAMPLE 17
[0344] This Example describes the further characterization of a
Peritrophin-like sequence cDNA, referred to herein as PL4, isolated
by EST sequencing described in Example 1.
[0345] A cDNA designated clone 2244-71 was isolated from the
unsubtracted HMT library as described in Example 1. Analysis of
clone 2244-71 indicated that the cDNA, denoted nCfPL4.sub.974, is
about 974 nucleotides in length, having a coding strand with
nucleic acid sequence SEQ ID NO:1892 and a complementary sequence
having SEQ ID NO:1893. Translation of SEQ ID NO:1892 suggests that
nucleic acid molecule nCfPL4.sub.974 encodes a partial-length
Peritrophin-like protein of 285 amino acids. Additional sequence
corresponding to the 5' end was isolated by PCR using the RACE cDNA
pool described in Example 3 as the template, as follows. Adapter
Primer 1, i.e. SEQ ID NO:1933, was used as the forward primer in
conjunction with reverse primer PL4-R1, which is complementary to
nucleotides 229-251 of SEQ ID NO:1892, having a nucleic acid
sequence 5' GAT ATC CAC TTT GAT CAG CGC AC 3', denoted herein as
SEQ ID NO:1946 in a PCR reaction under standard PCR reaction
conditions and the following thermocycling conditions: (1)
94.degree. C. for 30 seconds, (2) 5 cycles of 94.degree. C. for 10
seconds and 72.degree. C. for 4 minutes, (3) 5 cycles of 94.degree.
C. for 10 seconds and 70.degree. C. for 4 minutes, (4) 25 cycles of
94.degree. C. for 10 seconds then 68.degree. C. for 4 minutes. The
products of this reaction were diluted 1:50 and used as template in
a second PCR reaction using Adapter Primer 2, i.e. SEQ ID NO:1935
as the forward primer and reverse primer PL4-R2, which is
complementary to nucleotides 58-78 of SEQ ID NO:1892, having a
nucleic acid sequence 5' GGT ACT ACT CCT GGT GCG GGC 3', denoted
herein as SEQ ID NO:1947, using the thermocycling conditions
described for the first PCR reaction. The products of this reaction
were gel purified as previously described and the fragment was
ligated into the pCR II TA Cloning vector, available from Qiagen,
and sequenced to reveal of fragment of approximately 150
nucleotides in length. Sequence analysis revealed that nucleotides
68-146 of the fragment had 100% identity with nucleotides 1-79 of
nCfPL4.sub.974. The two sequences were aligned to form a contiguous
sequence of about 1043 nucleotides in length, referred to as
nCfPL4104.sub.3, having a coding strand with SEQ ID NO:1894 and a
complementary strand having SEQ ID NO:1895. However, the contiguous
sequence does not appear to encode a starting methionine in the
predicted protein sequence, thus, a second attempt to isolate the
remaining coding sequences at the 5' end was performed as follows.
A first PCR reaction was performed with Adapter Primer 1 as the
forward primer and PL4-R2 as the reverse primer using the RACE cDNA
pool as the template under the thermocycling conditions described
above. The products of this reaction were diluted 1:50 and used as
the template in a second PCR reaction which used Adapter Primer 2
as the forward primer and reverse primer PL4-R4, which is
complementary to nucleotides 58-80 of SEQ ID NO:1894, having the
nucleic acid sequence 5' CCG TCG ACA TTA AAC TCA CCA TC 3', denoted
SEQ ID NO:1948, under the thermocycling conditions described for
the first PCR reaction. The products of this reaction were gel
purified as previously described and the fragment was ligated into
the pCR II TA Cloning vector, available from Qiagen, and sequenced
to reveal of fragment of approximately 100 nucleotides in length.
Sequence analysis revealed that nucleotides 21-101 of the fragment
had 100% identity with nucleotides 1-81 of SEQ ID NO:1892. The two
sequences were aligned to form a contiguous sequence that is 1062
nucleotides in length, referred to herein as nCfPL4.sub.1062,
having a coding strand with SEQ ID NO:1896 and a complementary
strand with SEQ ID NO:1898. Translation of SEQ ID NO:1896 suggests
that nucleic acid molecule nCfPL4.sub.1062 encodes a full-length
Peritrophin-like protein of 285 amino acids, referred to herein as
PCfPL4.sub.285, having an amino acid sequence represented by SEQ ID
NO:1897, assuming the initiation codon spans from nucleotide 19
through nucleotide 21 of SEQ ID NO:1896 and the termination codon
spans from nucleotide 874 through nucleotide 876 of SEQ ID NO:1896.
The coding region encoding PCfPL4.sub.285, is represented by
nucleic acid molecule nCfPL4.sub.855, having a coding strand with
the nucleic acid sequence represented by SEQ ID NO:1899 and a
complementary strand with nucleic acid sequence represented by SEQ
ID NO:1900. The amino acid sequence of SEQ ID NO:1897, predicts
that PCfL4.sub.285 has an estimated molecular weight of about 31.4
kDa and an estimated isoelectric point (pI) of about 6.99.
[0346] Comparison of amino acid sequence SEQ ID NO:1897 with amino
acid sequences reported in GenBank indicates that SEQ ID NO:1897
showed the most homology, i.e., about 31.5% identity, with a
Drosophila melanogaster Gasp precourser (Accession #AAD09748).
Comparison of SEQ ID NO:1896 with nucleic acid sequences reported
in GenBank indicates that SEQ ID NO:1896 showed the most homology,
i.e., about 39.4% identity, with a Drosophila melanogaster Gasp
precourser (Accession #AF070734). Percent identity calculations
were performed using GCG version 9.0 using default parameters.
[0347] A Northern Blot analysis was conducted as described in
Example 4 to determine whether PL4 mRNA is expressed only in
certain life stages of the flea life cycle and whether PL4 mRNA is
expressed only in HMT tissue. Total RNA was extracted from eggs,
first, third, and wandering larvae, pupae, unfed adults, and adults
fed on cat blood for 0.25, 2, 8, and 24 hours. In addition, total
RNA was extracted from hindguts and Malpighian tubules extracted
from 24 hour cat blood-fed adult fleas, and from the remaining body
parts following the removal of hindguts and Malpighian tubules.
Each RNA sample was separated by gel electrophoresis, transferred
to nylon membranes and hybridized with .alpha.-.sup.32P-ATP labeled
nCfPL4.sub.974 under the Northern Blotting conditions described in
Example 4.
[0348] The results of the Northern blot assay are complex. Although
stringent conditions were used, several bands with distinct
expression patterns were seen. An approximately 1600 bp message was
detected in the egg, first instar, third instar and wandering
larval stages only. An approximately 1500 bp message was detected
in all lifestages and adult fed timepoints, but with the strongest
signals in the egg, first instar larval, and unfed adult stages. A
third message, which ran approximately 1200 bp, was detected in the
egg, first instar larval, pupal, and adult lifestages, including
all unfed and fed adult timepoints. All three of the messages
detected were seen only in the HMT tissues, and were not detected
in the carcass tissues.
[0349] The detection of three mRNAs instead of one may be the
result of the expression of three highly homologous transcripts. It
has been reported in the literature that peritrophin gene families
have been found that consist of a number of highly related genes
(See Schorderet et al., 1998, cDNA and deduced amino acid sequences
of a peritrophic membrane glycoprotein, `peritrophin-48`, from the
larvae of Lucilia cuprina Insect Biochemistry and Molecular Biology
28, 99-111). It is possible that these transcripts represent the
products of such a family or that the messages are the RNA products
of alternative splicing of a single gene locus.
EXAMPLE 18
[0350] This Example describes the further characterization of a
synaptic vesicle 2B-like sequence cDNA, isolated by EST sequencing
described in Example 1.
[0351] A cDNA designated clone 2104-59 was isolated from the
subtracted HMT library as described in Example 1, denoted herein as
SEQ ID NO:358. DNA from clone 2104-59 was purified, and the insert
used for plaque hybridization screening of the unsubtracted HMT
cDNA library as follows. The insert from clone 2104-59 was excised
by digestion with EcoRI, separated by agarose gel electrophoresis
and purified using the QiaQuick Gel Extraction kit, available from
Qiagen. A Megaprime DNA labeling kit, available from Amersham
Pharmacia, was used to incorporate .alpha.-.sup.32P-labeled dATP
into the random-primed probe mix. Hybridization and plaque
purification were performed as previously described which resulted
in the isolation of a clone containing an about 1875 nucleotide
synaptic vesicle 2B-like sequence, referred to herein as
nCfSVP.sub.1875, having a coding strand with nucleic acid sequence
SEQ ID NO:1901 and a complementary sequence having SEQ ID NO:1903.
Translation of SEQ ID NO:1901 suggests that nucleic acid molecule
nCfSVP.sub.1875 encodes a full-length synaptic vesicle 2B-like
protein of 530 amino acids, referred to herein as PCfSVP.sub.530,
having an amino acid sequence represented by SEQ ID NO:1902,
assuming the initiation codon spans from nucleotide 44 through
nucleotide 46 of SEQ ID NO:1901 and the termination codon spans
from nucleotide 1634 through nucleotide 1636 of SEQ ID NO:1901. The
coding region encoding PCfSVP.sub.530, is represented by nucleic
acid molecule nCfSVP.sub.1590, having a coding strand with the
nucleic acid sequence represented by SEQ ID NO:1904 and a
complementary strand with nucleic acid sequence represented by SEQ
ID NO:1905. The amino acid sequence of SEQ ID NO:1902, predicts
that PCfSVP.sub.530 has an estimated molecular weight of about 58.7
kDa and an pI of about 7.61.
[0352] Comparison of amino acid sequence SEQ ID NO:1902 with amino
acid sequences reported in GenBank indicates that SEQ ID NO:1902
showed the most homology, i.e., about 32% identity, with a
Drosophila melanogaster BACR7A4.y (Accession #CAB 51685).
Comparison of SEQ ID NO:1901 with nucleic acid sequences reported
in GenBank indicates that SEQ ID NO:1901 showed the most homology,
i.e., about 39% identity, with a Rattus norvegicus synaptic vesicle
protein 2B (SVP2B) mRNA (Accession #L10362). Percent identity
calculations were performed using GCG version 9.0 using default
parameters.
EXAMPLE 19
[0353] This Example describes the further characterization of a
Voltage-Gated Chloride Channel-like sequence cDNA, isolated by EST
sequencing described in Example 1.
[0354] A cDNA designated clone 2108-09 was isolated from the
unsubtracted HMT library as described in Example 1. Analysis of
clone 2108-09 indicated that the cDNA, denoted nCfVGCC.sub.381, is
about 381 nucleotides in length, having a coding strand with
nucleic acid sequence SEQ ID NO:1906 and a complementary sequence
having SEQ ID NO:1907. Translation of SEQ ID NO:1906 suggests that
nucleic acid molecule nCfVGCC.sub.381, encodes a partial-length
Voltage-Gated Chloride Channel-like protein of 126 amino acids.
Additional sequence corresponding to the 5' end was isolated by
hybridization and PCR as follows.
[0355] The insert from clone 2108-09 was excised by digestion with
EcoRI, separated by agarose gel electrophoresis and purified using
the QiaQuick Gel Extraction kit, available from Qiagen. A Megaprime
DNA labeling kit, available from Amersham Pharmacia, was used to
incorporate .alpha.-.sup.32P-labeled dATP into the random-primed
probe mix. Hybridization and plaque purification were performed on
the unsubtracted HMT cDNA library as previously described which
resulted in the isolation of a clone containing an about 2191
nucleotide VGCC-like sequence, referred to herein as
nCfVGCC.sub.2191, having a coding strand with nucleic acid sequence
SEQ ID NO:1908 and a complementary sequence having SEQ ID NO:1909.
Translation of SEQ ID NO:1908 suggests that nucleic acid molecule
nCfVGCC.sub.2191 encodes a partial s VGCC-like protein of 595 amino
acids.
[0356] In order to isolate the remaining coding regions at the 5'
end, a PCR was performed using the RACE cDNA pool, prepared as
described in Example 3, as the template as follows. Adapter Primer
1 was used as the forward primer in conjunction with reverse primer
VGCC-R1, which is complementary to the nucleotides 1482-1503 of SEQ
ID NO:1908, having a nucleic acid sequence 5' CGA TCA TGC GTC TAG
CAT TGG C 3', denoted herein as SEQ ID NO:1949 under standard PCR
reaction conditions and the following thermocycling conditions: (1)
94.degree. C. for 30 seconds, (2) 5 cycles of 94.degree. C. for 10
seconds and 72.degree. C. for 4 minutes, (3) 5 cycles of 94.degree.
C. for 10 seconds and 70.degree. C. for 4 minutes, (4) 25 cycles of
94.degree. C. for 10 seconds and 68.degree. C. for 4 minutes. The
reaction products were separated on an agarose gel and a band
corresponding to an approximately 1970 nucleotide molecule was
isolated, purified using a Gel Purification Kit, available from
Qiagen, ligated into the pCR II TA cloning vector, available from
Invitrogen, and sequenced using an ABI PRISM 377 automatic DNA
Sequencer. Sequence analysis revealed an approximately 1968
nucleotide fragment, referred to as nCfVGCC.sub.1968, having a
coding strand with SEQ ID NO:1910 and a complementary strand with
SEQ ID NO:1911. Sequence analysis also revealed that nucleic acid
molecule nCfVGCC.sub.1968 does not encode a start codon, thus, a
second 5' RACE PCR was performed as follows in order to isolate
additional sequence. Adapter Primer 1 was used as the forward
primer in conjunction with reverse primer VGCC-R4 primer which is
complementary to nucleotides 350-372 of SEQ ID NO:1910, having a
nucleic acid sequence 5' CCC GCC CCA GTT CTA GGT TGT CC 3', denoted
herein as SEQ ID NO:1950, using the RACE cDNA pool prepared as
described in Example 3 as the template, and the PCR reaction and
thermocycling conditions as described for the first PCR reaction.
The products of this reaction were then diluted 1:50 in water and
used as the template in a second PCR reaction with Adapter Primer 2
as the forward primer in conjunction with reverse primer VGCC-R2,
which is complementary to nucleotides 134-153 of SEQ ID NO:1910,
having a nucleic acid sequence 5' CAC ACC CAA CCT GAC CAG GC 3',
denoted herein as SEQ ID NO:1951, under the PCR reaction and
thermocycling conditions as described for the first PCR
reaction.
[0357] The products of this reaction were gel purified as
previously described and the fragment was ligated into the pCR II
TA Cloning vector, available from Qiagen, and sequenced to reveal
of fragment of approximately 673 nucleotides in length, referred to
herein as nCfVGCC.sub.673, having a coding strand with SEQ ID
NO:1912 and a complementary strand with SEQ ID NO:1913. Sequence
analysis revealed that nucleotides 520-673 of the fragment had 100%
identity with nucleotides 1-154 of SEQ ID NO:1910. The VGCC
fragments were aligned to form a contiguous sequence that is 3126
nucleotides in length, referred to herein as nCfVGCC.sub.3126,
having a coding strand with SEQ ID NO:1914 and a complementary
strand with SEQ ID NO:1916. Translation of SEQ ID NO:1914 suggests
that nucleic acid molecule nCfVGCC.sub.3126 encodes a full-length
VGCC-like protein of 851 amino acids, referred to herein as
PCfVGCC.sub.851, having an amino acid sequence represented by SEQ
ID NO:1915, assuming the initiation codon spans from nucleotide 168
through nucleotide 170 of SEQ ID NO:1914 and the termination codon
spans from nucleotide 2721 through nucleotide 2723 of SEQ ID
NO:1914. The coding region encoding PCfVGCC.sub.851 is represented
by nucleic acid molecule nCfVGCC.sub.2553, having a coding strand
with the nucleic acid sequence represented by SEQ ID NO:1917 and a
complementary strand with nucleic acid sequence represented by SEQ
ID NO:1918. The amino acid sequence of SEQ ID NO:1915, predicts
that PCfVGCC.sub.851 has an estimated molecular weight of about
93.4 kDa and an estimated pI of about 7.35.
[0358] Comparison of amino acid sequence SEQ ID NO:1915 with amino
acid sequences reported in GenBank indicates that SEQ ID NO:1915
showed the most homology, i.e., about 63.1% identity, with a
Oryctolagus cuniculus (rabbit) chloride channel protein 3 (CLCN3)
(Accession #AAB95163). Comparison of SEQ ID NO:1914 with nucleic
acid sequences reported in GenBank indicates that SEQ ID NO:1914
showed the most homology, i.e., about 61.3% identity, with a
Oryctolagus cuniculus chloride channel protein 3 (CLCN3) mRNA
(Accession #AF029348). Percent identity calculations were performed
using GCG version 9.0 using default parameters.
[0359] A Northern Blot analysis was conducted as described in
Example 4 to determine whether VGCC mRNA is expressed only in
certain life stages of the flea life cycle and whether VGCC mRNA is
expressed only in HMT tissue. Total RNA was extracted from eggs,
first, third, and wandering larvae, pupae, unfed adults, and adults
fed on cat blood for 0.25, 2, 8, and 24 hours. In addition, total
RNA was extracted from hindguts and Malpighian tubules extracted
from 24 hour cat blood-fed adult fleas, and from the remaining body
parts following the removal of hindguts and Malpighian tubules.
Each RNA sample was separated by gel electrophoresis, transferred
to nylon membranes and hybridized with .alpha.-.sup.32P-ATP labeled
nCfVGCC.sub.381 under the Northern Blotting conditions described in
Example 4. An approximately 3 kB band was detected in all
lifestages and adult unfed and fed timepoints, however, the
intensity of the signal did vary between stages with the strongest
signals seen in the egg, unfed adult, and 0.25 hour fed adult
stages, and the weakest signals seen in the 3rd instar larval and
pupal stages. A strong signal was detectable in the 24 hour fed
adult HMT tissues, but only a very weak signal was present in the
carcass tissues.
EXAMPLE 20
[0360] This Example describes the further characterization and
expression of an Intersectin-like cDNA isolated by EST sequencing
described in Example 2.
[0361] A cDNA designated clone 2225-23 was isolated from the
unsubtracted HNC library as described in Example 2, denoted herein
as SEQ ID NO:121. A Northern Blot analysis was conducted as
described in Example 10 to determine whether clone 2225-23 mRNA is
expressed exclusively in HNC tissues. For the hybridization step, a
probe comprising the flea clone 2225-23 nucleic acid molecule was
labeled with .alpha.-.sup.32P-ATP using a DNA labeling kit,
available from Amersham and added to the buffer at a concentration
of approximately 1.times.10.sup.6 cpm/ml, and allowed to hybridize
for about 14 to 18 hours at 42.degree. C. The blot was then washed
twice for 10minutes per wash in 0.5.times. SSPE and 0.1% sarcosyl
at 55.degree. C. and exposed to film for autoradiography. Analysis
of the developed film showed that there was greater expression of
clone 2225-23 mRNA in HNC tissues compared to non-HNC tissues,
indicating possible upregulation of clone 2225-23 in flea head and
nerve cords.
EXAMPLE 21
[0362] This Example describes the further characterization and
expression of an Neuroendocrine Specific Protein C-like cDNA
isolated by EST sequencing described in Example 2.
[0363] A cDNA designated clone 2249-19 was isolated from the
unsubtracted HNC library as described in Example 2, denoted herein
as SEQ ID NO:1775. A Northern Blot analysis was conducted as
described in Example 10 to determine whether clone 2249-19 mRNA is
expressed exclusively in HNC tissues. For the hybridization step, a
probe having the nucleic acid sequence of clone 2249-19 was
generated as follows. A PCR reaction was conducted using forward
primer 2249-19for, having a nucleotide sequence 5' AGT CGC ATA GTG
CAC TTC TGA ATG 3', denoted herein as SEQ ID NO:1954, and reverse
primer 2249-19rev, having a nucleotide sequence 5' CTG ACA TCT GTT
TCC ACA GCT C 3', denoted herein as SEQ ID NO:1955, using the HNC
cDNA library prepared as described in Example 2 as the template
under standard PCR reaction conditions and the following
thermocycling conditions: (1) one minute at 95.degree. C., (2) two
cycles of 94.degree. C. for 10 seconds, 50.degree. C. for 20
seconds, and 72.degree. C. for 20 seconds, (3) thirty cycles of
94.degree. C. for 10 sec, 53.degree. C. for 20 sec, 72.degree. C.
for 40 sec. The PCR product was ligated into the TA vector using a
TA cloning kit, available from Invitrogen and the clone was
digested with EcoRI enzyme, and purified from an agarose gel. The
purified nucleic acid molecule was labeled with
.alpha.-.sup.32P-ATP using a DNA labeling kit, available from
Amersham and added to the buffer at a concentration of
approximately 1.times.10.sup.6 cpm/ml, and allowed to hybridize for
about 14 to 18 hours at 42.degree. C. The blot was then washed
twice for 10 minutes per wash in 0.5.times. SSPE and 0.1% sarcosyl
at 55.degree. C. and exposed to film for autoradiography. Analysis
of the developed film showed that there was expression of clone
2249-19 mRNA in HNC tissues and non-HNC tissues with 2 bands
evident; one at approximately 1.5 Kb and one at approximately 2.5
Kb.
EXAMPLE 22
[0364] This Example describes the further characterization and
expression of an anoxia upregulated protein-like cDNA isolated by
EST sequencing described in Example 2.
[0365] A TA clone from the HNC EST library described in Example 2
designated clone 2218-95, denoted herein as SEQ ID NO:858 was
sequenced using standard sequencing methods and shown to contain a
non-full length nucleic acid molecule having significant homology
to anoxia upregulated protein (AUP) genes. Additional sequence
encoding an AUP gene was isolated as follows. A hybridization probe
containing the nucleic acid sequence of SEQ ID NO:1858 was
constructed as follows. A PCR reaction was conducted using forward
primer 2218-95for, having a nucleotide sequence 5' AAT AGT GAT GTT
GTA AGA GTT AGG 3', denoted herein as SEQ ID NO:1956, and reverse
primer 2218-95rev, having a nucleotide sequence 5' GTT TAA TAT TGC
ATG TTT ATT CAT TAA AA 3', denoted herein as SEQ ID NO:1957, using
the HNC cDNA library prepared as described in Example 2 as the
template under standard PCR reaction conditions and the following
thermocycling conditions: (1) one minute at 95.degree. C., (2)
thirty cycles of 94.degree. C. for 10 sec, 55.degree. C. for 20
sec, 72.degree. C. for 20 sec. The PCR product was ligated into the
TA vector using a TA cloning kit, available from Invitrogen and the
clone was digested with EcoRI enzyme, and purified from an agarose
gel. The purified nucleic acid molecule was labeled with
.alpha.-.sup.32P-ATP using a DNA labeling kit, available from
Amersham.
[0366] The .sup.32P .alpha.-dATP labeled probe was used in a
standard plaque lift hybridization procedure to isolate a clone
from the HNC lambda-ZAP unsubtracted cDNA library described in
Example 2. Hybridization was conducted as described in Example 12
and a plaque that hybridized strongly to the probe was isolated,
purified and sequenced as described in Example 12. Sequencing
revealed that the clone contained a nucleic acid molecule of about
1181 nucleotides, referred to herein as nCfAUP.sub.1181, having a
nucleotide sequence denoted herein as SEQ ID NO:1919. The
complement of SEQ ID NO:1919 is represented herein as SEQ ID
NO:1921.
[0367] Translation of SEQ ID NO:1919 suggests that nucleic acid
molecule nCfAUP.sub.1181 encodes a full-length AUP protein of 102
amino acids, referred to herein as PCfAUP.sub.102, having an amino
acid sequence represented by SEQ ID NO:1920, assuming the
initiation codon spans from nucleotide 127 through nucleotide 129
of SEQ ID NO:1919 and the termination codon spans from nucleotide
433 through nucleotide 435 of SEQ ID NO:1919. The coding region
encoding PCfAUP.sub.102, is represented by nucleic acid molecule
nCfAUP.sub.306, having a coding strand with the nucleic acid
sequence represented by SEQ ID NO:1922 and a complementary strand
with nucleic acid sequence represented by SEQ ID NO:1923. The amino
acid sequence of PCfAUP.sub.102, predicts that PCfAUP.sub.102 has
an estimated molecular weight of about 11.9 kDa and an estimated pI
of about 10.5.
[0368] Comparison of amino acid sequence SEQ ID NO:1920 with amino
acid sequences reported in GenBank indicates that SEQ ID NO:1920
showed the most homology, i.e., about 52% identity, with a
Drosophila melanogaster anoxia upregulated protein, GenBank
Accession No. AAD38397. Percent identity calculations were
performed using GCG version 9.0 using default parameters. Blast
comparison of nucleic acid sequence SEQ ID NO:1919 with nucleic
acid sequences reported in GenBank indicates that SEQ ID NO:1919
showed the most homology to a clone from human chromosome 14q31
region containing gene for neurexin III, GenBank #AC007056.
Pairwise identity could not be performed as the human clone in
GenBank is too large to load into GCG version 9.0.
EXAMPLE 23
[0369] This Example describes the further characterization of a
neuroendocrine specific 7B2 polypeptide, isolated by EST sequencing
described in Example 2.
[0370] A cDNA designated clone 2211-21 was isolated from the
subtracted HNC library as described in Example 2, denoted herein as
SEQ ID NO:92. DNA from clone 2211-21 was purified, and the insert
used for plaque hybridization screening of the unsubtracted HMT
cDNA library as follows. The insert from clone 2211-21 was excised
by digestion with EcoRI, separated by agarose gel electrophoresis
and purified using the QiaQuick Gel Extraction kit, available from
Qiagen. A Megaprime DNA labeling kit, available from Amersham
Pharmacia, was used to incorporate .alpha.-.sup.32P-labeled dATP
into the random-primed probe mix. The .sup.32P .alpha.-dATP labeled
probe was used in a standard plaque lift hybridization procedure to
isolate a clone from the HNC lambda-ZAP unsubtracted cDNA library,
prepared as described in Example 2. The following hybridization
conditions were used. Hybond-N filters, available from Amersham,
were hybridized with about 2.times.10.sup.6 counts per minute (cpm)
per ml of the probe in 50 ml of hybridization solution (5.times.
SSPE, 25mM EDTA pH 8.0, 5.times. Denhardt's reagent, 1.2% SDS,
0.020 mg/mL salmon sperm DNA) at 55.degree. C. for about 48 hours.
The filters were washed once in 50 mL 4.times. SSPE, 1% SDS for 15
minutes at 55.degree. C., once in 50 mL 2.times. SSPE, 1% SDS for
10 minutes at 55.degree. C., and washed twice in 50 mL 0.5.times.
SSPE, 0.5% SDS for 10 minutes at 55.degree. C. The filters were
then subjected to autoradiography. One plaque that hybridized
strongly to the probe was isolated and subjected to in vivo
excision using the Stratagene Ex-Assist.TM. helper phage system and
protocols. Miniprep DNA was prepared from the positive clone using
a Miniprep kit and protocol, available from Qiagen, Chatsworth,
Calif., and sequenced using standard sequencing procedures. The
clone, referred to as nCf7B2.sub.2161 contains a nucleic acid
molecule of about 2161 nucleotides in length, having a coding
strand with nucleic acid sequence SEQ ID NO:1924 and a
complementary sequence having SEQ ID NO:1926.
[0371] Translation of SEQ ID NO:1924 suggests that nucleic acid
molecule nCf7B2.sub.2161 encodes a full-length 7B2-like protein of
267 amino acids, referred to herein as PCf7B2.sub.267, having an
amino acid sequence represented by SEQ ID NO:1925, assuming the
initiation codon spans from nucleotide 107 through nucleotide 109
of SEQ ID NO:1924 and the termination codon spans from nucleotide
908 through nucleotide 910 of SEQ ID NO:1924. The coding region
encoding PCf7B2.sub.267, is represented by nucleic acid molecule
nCf7B2.sub.801, having a coding strand with the nucleic acid
sequence represented by SEQ ID NO:1927 and a complementary strand
with nucleic acid sequence represented by SEQ ID NO:1928. The amino
acid sequence of SEQ ID NO:1925, predicts that PCf7B2.sub.267 has
an estimated molecular weight of about 31 kDa and an estimated pI
of about 5. Analysis of PCf7B2.sub.267 suggests the presence of a
signal peptide encoded by a stretch of amino acids spanning from
about amino acid 1 through amino acid 20. The proposed mature
protein, referred to herein as PCf7B2.sub.247 contains 247 amino
acids, designated SEQ ID NO:1930, and is encoded by a nucleic acid
molecule referred to as nCf7B2.sub.741, having a coding strand with
SEQ ID NO:1929 and a complementary strand with SEQ ID NO:1931.
[0372] Comparison of amino acid sequence SEQ ID NO:1925 with amino
acid sequences reported in GenBank indicates that SEQ ID NO:1925
showed the most homology, i.e., about 39% identity, with a
Drosophila melanogaster protein, GenBank Accession No. AAF52036.
Percent identity calculations were performed using GCG version 9.0
using default parameters. Blast comparison of nucleic acid sequence
SEQ ID NO:1924 with nucleic acid sequences reported in GenBank
indicates that SEQ ID NO:1924 showed the most homology to a human
chromosome 19, cosmid R28204 clone, GenBank #Accession No.
AC006132. Pairwise identity could not be performed as the human
clone in GenBank is too large to load into GCG version 9.0,
however, the BLAST score was 0.20, which is not considered to be
significant level of identity.
[0373] A Northern Blot analysis was conducted as described in
Example 10 to determine whether 7B2 mRNA is expressed exclusively
in HNC tissues. For the hybridization step, a probe having the
nucleic acid sequence of clone 2211-21 was generated as follows. A
PCR reaction was conducted using forward primer 2211-21for, having
a nucleotide sequence 5' GCG CCA TGA AGA TTT CAG GCG 3', denoted
herein as SEQ ID NO:1958, and reverse primer 2211-21rev, having a
nucleotide sequence 5' AAG TGC AAT GAA TCA TCA GCA AG 3', denoted
herein as SEQ ID NO:1959, using the HNC cDNA library prepared as
described in Example 2 as the template under standard PCR reaction
conditions and the following thermocycling conditions: (1) one
minute at 95.degree. C., (2) five cycles of 94.degree. C. for 10
seconds, 50.degree. C. for 20 seconds, and 72.degree. C. for 20
seconds, (3) thirty cycles of 94.degree. C. for 10 sec, 53.degree.
C. for 20 sec, 72.degree. C. for 40 sec. The PCR product was
ligated into the TA vector using a TA cloning kit, available from
Invitrogen and the clone was digested with EcoRI enzyme, and
purified from an agarose gel. The purified nucleic acid molecule
was labeled with .alpha.-.sup.32P-ATP using a DNA labeling kit,
available from Amersham and added to the buffer at a concentration
of approximately 1.times.10.sup.6 cpm/ml, and allowed to hybridize
for about 14 to 18 hours at 42.degree. C. The blot was then washed
twice for 10 minutes per wash in 0.5.times. SSPE and 0.1% sarcosyl
at 55.degree. C. and exposed to film for autoradiography. Analysis
of the developed film showed that after 2.5 days of exposure clone
2211-21 mRNA was expressed exclusively in HNC tissue.
[0374] While various embodiments of the present invention have been
described in detail, it is apparent that modifications and
adaptations of those embodiments will occur to those skilled in the
art. It is to be expressly understood, however, that such
modifications and adaptations are within the scope of the present
invention, as set forth in the following claims:
Sequence CWU 0
0
* * * * *