U.S. patent application number 10/678521 was filed with the patent office on 2004-04-29 for novel carboxylesterase nucleic acid molecules, proteins and uses thereof.
Invention is credited to Brandt, Kevin S., Silver, Gary M., Wisnewski, Nancy.
Application Number | 20040081998 10/678521 |
Document ID | / |
Family ID | 25004163 |
Filed Date | 2004-04-29 |
United States Patent
Application |
20040081998 |
Kind Code |
A1 |
Silver, Gary M. ; et
al. |
April 29, 2004 |
Novel carboxylesterase nucleic acid molecules, proteins and uses
thereof
Abstract
The present invention relates to arthropod esterase proteins; to
arthropod esterase nucleic acid molecules, including those that
encode such esterase proteins; to antibodies raised against such
esterase proteins; and to other compounds that inhibit arthropod
esterase 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 such proteins, nucleic acid
molecules, antibodies and/or inhibitory compounds as well as the
use of such therapeutic compositions to protect animals from
hematophagous arthropod infestation.
Inventors: |
Silver, Gary M.; (Fort
Collins, CO) ; Wisnewski, Nancy; (Fort Collins,
CO) ; Brandt, Kevin S.; (Ft. Collins, CO) |
Correspondence
Address: |
HESKA CORPORATION
INTELLECTUAL PROPERTY DEPT.
1613 PROSPECT PARKWAY
FORT COLLINS
CO
80525
US
|
Family ID: |
25004163 |
Appl. No.: |
10/678521 |
Filed: |
October 2, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10678521 |
Oct 2, 2003 |
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09403942 |
May 2, 2000 |
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6664090 |
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09403942 |
May 2, 2000 |
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PCT/US97/20598 |
Nov 10, 1997 |
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09403942 |
May 2, 2000 |
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08747221 |
Nov 12, 1996 |
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6063610 |
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Current U.S.
Class: |
435/6.13 ;
435/196; 435/320.1; 435/348; 435/69.1; 530/388.26; 536/23.2 |
Current CPC
Class: |
A61P 33/12 20180101;
A61K 38/00 20130101; C12N 9/18 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/196; 435/348; 435/320.1; 530/388.26; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/16; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated nucleic acid molecule that hybridizes under
stringent hybridization conditions with a gene 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:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ
ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27,
SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID
NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ
ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60,
SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID
NO:71, SEQ ID NO:72, SEQ ID NO:76 and a nucleic acid molecule
encoding a protein comprising amino acid sequence SEQ ID NO:74.
2. An isolated nucleic acid molecule that hybridizes under
stringent hybridization conditions with a nucleic acid molecule
having a nucleic acid sequence encoding a protein comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:1, SEQ ID NO:14, SEQ ID
NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ
ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44,
SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID
NO:68, SEQ ID NO:73 and SEQ ID NO:74.
3. An isolated 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:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,
SEQ ID NO:23, SEQ ID NO:24, SEQ. ID NO:26, SEQ ID NO:27, SEQ ID
NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ
ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51,
SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID
NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ
ID NO:72, SEQ ID NO:76 a nucleic acid molecule encoding a protein
comprising amino acid sequence SEQ ID NO:74; and a nucleic acid
molecule comprising an allelic variant of a nucleic acid molecule
comprising any of said nucleic acid sequences.
4. An isolated protein encoded by a nucleic acid molecule that
hybridizes under stringent hybridization conditions to a nucleic
acid molecule selected from the group consisting of: a nucleic acid
molecule comprising 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:17, SEQ ID NO:20, SEQ ID NO:22, SEQ ID
NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ
ID NO:52, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:69, SEQ ID NO:71;
and a nucleic acid molecule encoding a protein comprising an amino
acid sequence selected from the group consisting of SEQ ID NO:39,
SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID
NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55 and SEQ ID
NO:74.
5. An isolated flea protein selected from the group consisting of:
a protein comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11,
SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID
NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ
ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55,
SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73 and SEQ ID NO:74; and a
protein encoded by an allelic variant of a nucleic acid molecule
encoding a protein comprising any of said amino acid sequences.
6. A formulation of flea carboxylesterase proteins, wherein said
proteins, when submitted to 14% Tris-glycine SDS-PAGE, comprise a
fractionation profile as depicted in FIG. 3, wherein said proteins
have carboxylesterase activity.
7. A formulation of flea carboxylesterase proteins, wherein said
proteins, when submitted to IEF-PAGE, comprise a fractionation
profile as depicted in FIG. 4 in a lane selected from the group
consisting of lane 3, lane 4, lane 5, lane 6 and lane 7, wherein
said proteins have carboxylesterase activity.
8. An isolated flea protein that hydrolyzes .alpha.-napthyl acetate
to produce .alpha.-napthyl, when said protein is incubated in the
presence of .alpha.-napthyl acetate contained in about 20 mM Tris
at about pH 8.0 for about 15 minutes at about 37.degree. C.
9. A formulation comprising flea proteins that hydrolyze
.alpha.-napthyl acetate to produce .alpha.-napthol, when said
proteins are incubated in the presence of .alpha.-napthyl acetate
contained in about 20 mM Tris at about pH 8.0 for about 15 minutes
at about 37.degree. C.
10. An isolated flea protein that hydrolyzes the methyl ester group
of juvenile hormone to produce a juvenile hormone acid.
11. A formulation comprising flea proteins that hydrolyze the
methyl ester group of juvenile hormone to produce a juvenile
hormone acid.
12. A therapeutic composition that, when administered to an animal,
reduces hematophagous ectoparasite infestation, said therapeutic
composition comprising an excipient and a protective compound
selected from the group consisting of: an isolated hematophagous
ectoparasite carboxylesterase protein; a mimetope of an isolated
hematophagous ectoparasite carboxylesterase protein; an isolated
hematophagous ectoparasite carboxylesterase nucleic acid molecule
that hybridizes under stringent hybridization conditions with a
Ctenocephalides felis carboxylesterase gene; an isolated antibody
that selectively binds to a hematophagous ectoparasite
carboxylesterase protein; and an inhibitor of carboxylesterase
activity identified by its ability to inhibit the activity of a
flea carboxylesterase.
13. A method to reduce hematophagous ectoparasite infestation
comprising treating an animal with a therapeutic composition
comprising a protective compound selected from the group consisting
of: an isolated hematophagous ectoparasite carboxylesterase
protein; a mimetope of a hematophagous ectoparasite
carboxylesterase protein; an isolated hematophagous ectoparasite
carboxylesterase nucleic acid molecule that hybridizes under
stringent hybridization conditions with a Ctenocephalides felis
carboxylesterase gene; an isolated antibody that selectively binds
to a hematophagous ectoparasite carboxylesterase protein; and an
inhibitor of carboxylesterase activity identified by its ability to
inhibit the activity of a flea carboxylesterase.
14. A method to produce a carboxylesterase protein, said method
comprising culturing a cell capable of expressing said protein,
said protein being encoded by a nucleic acid molecule that
hybridizes under stringent hybridization conditions with a
Ctenocephalides felis carboxylesterase gene.
15. A method to identify a compound capable of inhibiting flea
carboxylesterase activity, said method comprising: (a) contacting
an isolated flea carboxylesterase with a putative inhibitory
compound under conditions in which, in the absence of said
compound, said protein has carboxylesterase activity; and (b)
determining if said putative inhibitory compound inhibits said
activity.
16. A test kit to identify a compound capable of inhibiting flea
carboxylesterase activity, said test kit comprising an isolated
flea carboxylesterase protein having esterase activity and a means
for determining the extent of inhibition of said activity in the
presence of a putative inhibitory compound.
17. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule is a flea nucleic acid molecule.
18. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule is selected from the group consisting of Ctenocephalides,
Ceratophyllus, Diamanus, Echidnophaga, Nosopsyllus, Pulex, Tunga,
Oropsylla, Orchopeus and Xenopsylla nucleic acid molecules.
19. The nucleic acid molecule of claims 1 or 2, wherein said
nucleic acid molecule is selected from the group consisting of
Ctenocephalides felis, Ctenocephalides canis, Ceratophyllus
pulicidae, Pulex irritans, Oropsylla (Thrassis) bacchi, Oropsylla
(Diamanus) montana, Orchopeus howardi, Xenopsylla cheopis and Pulex
simulans nucleic acid molecules.
20. The nucleic acid molecule of claims 1 or 2, wherein said
nucleic acid molecule comprises a Ctenocephalides felis nucleic
acid molecule.
21. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule hybridizes under stringent hybridization conditions with a
nucleic acid molecule selected from the group consisting of
nfE1.sub.401, nfE2.sub.364, nfE3.sub.421, nfE4.sub.524,
nfE5.sub.1982, nfE5.sub.1515, nfE5.sub.2144, nfE5.sub.1650,
nfE6.sub.1488, nfE6.sub.1792, nfE6.sub.1650, nfE7.sub.2836,
nfE7.sub.1788, nfE7.sub.1710, nfE7.sub.650, nfE8.sub.2801,
nfE8.sub.1785, nfE8.sub.1710, nfE9.sub.2007, nfE9.sub.1584,
nfE9.sub.1540, nfE10.sub.1987 and nfE10.sub.1590.
22. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule comprises a nucleic acid molecule selected from the group
consisting of nfE1.sub.401, nfE2.sub.364, nfE3.sub.421,
nfE4.sub.524, nfE5.sub.1982, nfE5.sub.1515, nfE5.sub.2144,
nfE5.sub.1650, nfE6.sub.1488, nfE6.sub.1792, nfE6.sub.1650,
nfE7.sub.2836, nfE7.sub.1788, nfE7.sub.1710, nfE7.sub.650,
nfE8.sub.2801, nfE8.sub.1785, nfE8.sub.1710, nfE9.sub.2007,
nfE9.sub.1584, nfE9.sub.1540, nfE10.sub.1987 and nfE10.sub.590.
23. The nucleic acid molecule of claims 1 or 2, wherein said
nucleic acid molecule is selected from the group consisting of: a
nucleic acid molecule comprising a nucleic acid sequence that
encodes a protein having an amino acid sequence selected from the
group consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID
NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ
ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,
SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID
NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73 and SEQ ID NO:74;
and a nucleic acid molecule comprising an allelic variant of a
nucleic acid molecule encoding a protein having any of said amino
acid sequences.
24. The nucleic acid molecule of claims 1 or 2, 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:17, SEQ ID
NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ
ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,
SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID
NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ
ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:69,
SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76, a nucleic
acid molecule encoding a protein comprising amino acid sequence SEQ
ID NO:74 SEQ ID NO:74; and a nucleic acid molecule comprising an
allelic variant of a nucleic acid molecule comprising any of said
nucleic acid sequences.
25. The nucleic acid molecule of claim 1, wherein said nucleic acid
molecule comprises an oligonucleotide.
26. A recombinant molecule comprising a nucleic acid molecule as
set forth in claims 1 or 2 operatively linked to a transcription
control sequence.
27. A recombinant virus comprising a nucleic acid molecule as set
forth in claims 1 or 2.
28. A recombinant cell comprising a nucleic acid molecule as set
forth in claims 1 or 2.
29. The nucleic acid molecule of claim 2, wherein said nucleic acid
molecule comprises a nucleic acid sequence that encodes a
carboxylesterase protein.
30. The nucleic acid molecule of claim 2, wherein said nucleic acid
molecule hybridizes under stringent hybridization conditions with
the complement of a nucleic acid sequence encoding said
protein.
31. The protein of claim 4, wherein said protein, when administered
to an animal, elicits an immune response against a carboxylesterase
protein.
32. The protein of claim 4, wherein said protein is encoded by a
nucleic acid molecule that hybridizes under stringent hybridization
conditions with nucleic acid molecule nfE6.sub.1792.
33. The protein of claim 4, wherein said protein is selected from
the group consisting of: a protein comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:2, SEQ ID
NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID
NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ
ID NO:41. SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53,
SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58 SEQ ID NO:68, SEQ ID NO:73
and SEQ ID NO:74; and a protein encoded by an allelic variant of a
nucleic acid molecule encoding a protein selected from the group
consisting of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11,
SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID
NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ
ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55,
SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73 and SEQ ID NO:74.
34. An isolated antibody that selectively binds to a protein as set
forth in claim 4.
35. The formulation of claim 6, wherein said proteins are selected
from the group consisting of proteins having molecular weights
ranging from about 60 kD to about 75 kD as determined by 14%
Tris-glycine SDS-PAGE.
36. The formulation of claim 6, wherein said proteins are isolated
by the method comprising: (a) applying soluble proteins of a flea
extract to a p-aminobenzamidine agarose bead column; (b) collecting
unbound protein from said p-aminobenzamidine agarose bead column
and applying said unbound protein to a gel filtration column; (c)
eluting proteins bound to said gel filtration column and applying
said eluted proteins to a cation exchange chromatography column;
(d) eluting proteins bound to said cation exchange column and
applying said eluted proteins to an anion exchange chromatography
column; and (e) eluting proteins bound to said anion exchange
column with about 170 mM NaCl to obtain said flea carboxylesterase
proteins.
37. The formulation of claim 7, wherein said proteins are selected
from the group consisting of proteins having pI values ranging from
about pI 4.7 to about pI 5.2 as determined by IEF-PAGE.
38. The invention of claims 8 or 9, wherein said incubation results
in the production of from about 0.3 to at least about 2.5
absorbance units in the presence of Fast Blue when measured at 590
nm.
39. The protein of claim 8, wherein said protein does not hydrolyze
the methyl ester group of juvenile hormone.
40. The invention of claims 8 or 9 or 10 or 11, wherein the mature
form of said protein has a molecular weight ranging from about 60
kD to about 75 kD.
41. The invention of claims 8 or 9, wherein said protein comprises
an amino acid sequence selected from the group consisting of SEQ ID
NO:5, SEQ ID NO:19, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ
ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and SEQ ID NO:53.
42. The invention of claims 10 or 11, wherein hydrolyzation of said
methyl ester group results in the release of at least about 120
counts per minute of .sup.3H-juvenile hormone acid when determined
by the method comprising: (a) incubating said flea protein in the
presence of .sup.3H-juvenile hormone to create a reaction mixture;
(b) combining said reaction mixture with isooctane to produce an
aqueous phase and an organic phase; (c) recovering said aqueous
phase; and (d) determining the amount of .sup.3H-juvenile hormone
acid present in said aqueous phase.
43. The protein of claim 10, wherein said protein does not
hydrolyze .alpha.-napthyl acetate.
44. The invention of claims 12 or 13, wherein said hematophagous
ectoparasite carboxylesterase comprises a flea
carboxylesterase.
45. The invention of claims 12 or 13, wherein said hematophagous
ectoparasite is a flea of a genus selected from the group
consisting of Ctenocephalides, Ceratophyllus, Diamanus,
Echidnophaga, Nosopsyllus, Pulex, Tunga, Oropsylla, Orchopeus and
Xenopsylla.
46. The invention of claims 12 or 13, wherein said hematophagous
ectoparasite is a flea of a species selected from the group
consisting of Ctenocephalides felis, Ctenocephalides canis,
Ceratophyllus pulicidae, Pulex irritans, Oropsylla (Thrassis)
bacchi, Oropsylla (Diamanus) montana, Orchopeus howardi, Xenopsylla
cheopis and Pulex simulans.
47. The invention of claims 12 or 13, wherein said hematophagous
ectoparasite carboxylesterase comprises a juvenile hormone
esterase.
48. The invention of claims 12 or 13, wherein said composition
further comprises a component selected from the group consisting of
an adjuvant and a carrier.
49. The invention of claims 12 or 13, wherein said composition
further comprises a compound that reduces hematophagous
ectoparasite burden by a method other than by reducing
hematophagous ectoparasite carboxylesterase activity.
50. The invention of claims 12 or 13, wherein said protective
compound is selected from the group consisting of a naked nucleic
acid vaccine, a recombinant virus vaccine and a recombinant cell
vaccine.
51. The invention of claims 12 or 13, wherein said inhibitor
comprises a substrate analog of a hematophagous ectoparasite
carboxylesterase.
52. The method of claim 13, wherein said animal is selected from
the group consisting of adult hematophagous ectoparasites,
hematophagous ectoparasite larvae and animals susceptible to
hematophagous ectoparasite infestation.
53. The method of claim 13, wherein said animal is selected from
the group consisting of adult fleas, flea larvae and animals
susceptible to flea infestation.
54. The method of claim 13, wherein hematophagous ectoparasite
infestation is reduced by hematophagous ectoparasite larvae
ingesting adult hematophagous ectoparasite feces comprising said
therapeutic composition.
55. The method of claim 13, wherein said adult hematophagous
ectoparasite feces comprises anti-hematophagous ectoparasite
carboxylesterase antibodies.
56. The method of claim 13, wherein anti-hematophagous ectoparasite
carboxylesterase antibodies are elicited in a host animal in
response to administration of a reagent selected from the group
consisting of one or more of said isolated hematophagous
ectoparasite carboxylesterase proteins and one or more of said
hematophagous ectoparasite carboxylesterase nucleic acid molecules,
said adult hematophagous ectoparasite having fed from said host
animal after said administration.
57. The method of claim 13, wherein said animal is selected from
the group consisting of mammals and birds.
58. The method of claim 13, wherein said animal is selected from
the group consisting of cats and dogs.
59. The method of claim 14, wherein said cell expresses a nucleic
acid molecule selected from the group consisting of
pCro-nfE6.sub.1488, pTrc-nfE7.sub.650, pTrc-nfE7.sub.1710,
pTrc-nfE8.sub.1710, pTrc-nfE5.sub.1650, pTrc-nfE9.sub.1540,
pFB-nfE6.sub.1679, pVL-nfE7.sub.1802, pVL-fE8.sub.1792 and
pVL-nfE9.sub.1600.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to arthropod esterase nucleic
acid molecules, 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 their use to
protect an animal from hematophagous arthropod infestation.
BACKGROUND OF THE INVENTION
[0002] Hematophagous arthropod infestation of animals is a health
and economic concern because hematophagous arthropods are known to
cause and/or transmit a variety of diseases. Hematophagous
arthropods 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 hematophagous arthropods 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,
hematophagous arthropods are a problem not only when they are on an
animal but also when they are in the general environment of the
animal.
[0003] Bites from hematophagous arthropods 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.
[0004] The medical importance of arthropod infestation has prompted
the development of reagents capable of controlling arthropod
infestation. Commonly encountered methods to control arthropod
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 arthropod populations.
In particular, insecticides have been used to prevent hematophagous
arthropod infestation of animals by adding such insecticides to
shampoos, powders, collars, sprays, foggers and liquid bath
treatments (i.e., dips). Reduction of hematophagous arthropod
infestation on the pet has been unsuccessful for one or more of the
following reasons: (1) failure of owner compliance (frequent
administration is required); (2) behavioral or physiological
intolerance of the pet to the pesticide product or means of
administration; and (3) the emergence of hematophagous arthropod
populations resistant to the prescribed dose of pesticide. However,
hematophagous arthropod populations have been found to become
resistant to insecticides.
[0005] Prior investigators have described insect carboxylesterase
(CE) protein biochemistry, for example, Chen et al., Insect
Biochem. Molec. Biol., 24:347-355, 1994; Whyard et al., Biochemical
Genetics, 32:924, 1994 and Argentine et al., Insect Biochem. Molec
Biol, 25:621-630, 1995. Other investigators have disclosed certain
insect CE amino acid sequences, for example, Mouches et al., Proc
Natl Acad Sci USA, 87:2574-2578, 1990 and Cooke et al., Proc Natl
Acad Sci USA, 86:1426-1430, 1989, and nucleic acid sequence (Vaughn
et al., J. Biol. Chem., 270:17044-17049, 1995).
[0006] Prior investigators have described certain insect juvenile
hormone esterase (JHE) nucleic acid and amino acid sequences: for
example, sequence for Heliothis virescens is disclosed by Hanzlik
et al., J. Biol. Chem., 264:12419-12425, 1989; Eldridge et al., App
Environ Microbiol, 58:1583-1591, 1992; Bonning et al., Insect
Biochem. Molec. Biol., 22:453-458, 1992; Bonning et al., Natural
and Engineered Pest Management Agents, pp. 368-383, 1994 and
Harshman et al., Insect Biochem. Molec. Biol, 24:671-676, 1994;
sequence for Manduca sexta-s disclosed by Vankatesh et al., J Biol
Chem, 265:21727-21732, 1990; sequence for Trichopulsia ni is
disclosed by Venkataraman et al., Dev. Genet., 15:391-400, 1994 and
Jones et al., Biochem. J., 302:827-835, 1994; and sequence for
Lymantria dispar is disclosed by Valaitis, Insect Biochem. Molec.
Biol., 22:639-648, 1992.
[0007] Identification of an esterase of the present invention is
unexpected, however, because even the most similar nucleic acid
sequence identified by previous investigators could not be used to
identify an esterase of the present invention. In addition,
identification of an esterase protein of the present invention is
unexpected because a protein fraction from flea prepupal larvae
that was obtained by monitoring for serine protease activity
surprisingly also contained esterase proteins of the present
invention.
[0008] In summary, there remains a need to develop a reagent and a
method to protect animals or plants from hematophagous arthropod
infestation.
SUMMARY OF THE INVENTION
[0009] The present invention relates to a novel product and process
for protection of animals or plants from arthropod infestation.
According to the present invention there are provided arthropod
esterase proteins and mimetopes thereof; arthropod nucleic acid
molecules, including those that encode such proteins; antibodies
raised against such esterase proteins (i.e., anti-arthropod
esterase antibodies); and compounds that inhibit arthropod esterase
activity (i.e, inhibitory compounds or inhibitors).
[0010] The present invention also includes methods to obtain such
proteins, mimetopes, nucleic acid molecules, antibodies and
inhibitory compounds. Also included in the present invention are
therapeutic compositions comprising such proteins, mimetopes,
nucleic acid molecules, antibodies, and/or inhibitory compounds, as
well as use of such therapeutic compositions to protect animals
from arthropod infestation.
[0011] Identification of an esterase of the present invention is
unexpected, however, because the most similar nucleic acid sequence
identified by previous investigators could not be used to identify
an esterase of the present invention. In addition, identification
of an esterase protein of the present invention is unexpected
because a protein fraction from flea prepupal larvae that was
obtained by monitoring for serine protease activity surprisingly
also contained esterase proteins of the present invention.
[0012] One embodiment of the present invention is an isolated
nucleic acid molecule that hybridizes under stringent hybridization
conditions with a gene comprising a nucleic acid sequence including
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:17, SEQ ID NO:18, SEQ ID NO:20, SEQ
ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26,
SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID
NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ
ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59,
SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID
NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic
acid molecule encoding a protein comprising amino acid sequence SEQ
ID NO:74.
[0013] The present invention also includes a nucleic acid molecule
that hybridizes under stringent hybridization conditions with a
nucleic acid molecule encoding a protein comprising at least one of
the following amino acid sequences:SEQ ID NO:2, SEQ ID NO:5, SEQ ID
NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ
ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41,
SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID
NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73
and/or SEQ ID NO:74; and particularly a nucleic acid molecule that
hybridizes with a nucleic acid sequence that is a complement of a
nucleic acid sequence encoding any of the amino acid sequences. A
preferred nucleic acid molecule of the present invention includes a
nucleic acid molecule comprising a nucleic acid sequence including
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:17, SEQ ID NO:18, SEQ ID NO:20, SEQ
ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26,
SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID
NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ
ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59,
SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID
NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic
acid molecule encoding a protein comprising amino acid sequence SEQ
ID NO:74, and allelic variants thereof.
[0014] The present invention also includes an isolated
carboxylesterase nucleic acid molecule comprising a nucleic acid
sequence encoding a protein comprising an amino acid sequence
including SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:39, SEQ ID NO:40,
SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and/or SEQ
ID NO:53. SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,
SEQ ID NO:43 and SEQ ID NO:44 represent N-terminal amino acid
sequences of carboxylesterases isolated from prepupal flea larvae,
the production of which are described in the Examples of the
present application.
[0015] 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.
[0016] Another embodiment of the present invention includes an
isolated esterase protein that is encoded by a nucleic acid
molecule that hybridizes under stringent hybridization conditions
to (a) a nucleic acid molecule that includes at least one of the
following nucleic acid sequences:SEQ ID NO:3, SEQ ID NO:6, SEQ ID
NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:20, SEQ
ID NO:22, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35,
SEQ ID NO:38, SEQ ID NO:52, SEQ ID NO:59, SEQ ID NO:61, SEQ ID
NO:69, and SEQ ID NO:71; and/or (b) a nucleic acid molecule
encoding a protein including at least one of the following amino
acid sequences: SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID
NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ
ID NO:55 and SEQ ID NO:74. One embodiment is a carboxylesterase
protein encoded by a nucleic acid molecule that hybridizes under
stringent hybridization conditions to a nucleic acid molecule that
encodes a protein comprising at least one of the following amino
acid sequences: SEQ ID NO:5, SEQ ID NO:19; SEQ ID NO:39, SEQ ID
NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44
and/or SEQ ID NO:53. Preferred proteins of the present invention
are isolated flea proteins including at least one of the following
amino acid sequences:SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID
NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ
ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42,
SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53, SEQ ID NO:54, SEQ ID
NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID NO:73 and SEQ ID NO:74;
also included are proteins encoded by allelic variants of nucleic
acid molecules encoding proteins comprising any of the above-listed
amino acid sequences.
[0017] The present invention also relates to mimetopes of arthropod
esterase proteins as well as to isolated antibodies that
selectively bind to arthropod esterase proteins or mimetopes
thereof. Also included are methods, including recombinant methods,
to produce proteins, mimetopes and antibodies of the present
invention.
[0018] The present invention also includes a formulation of flea
carboxylesterase proteins, in which the proteins, when submitted to
14% Tris-glycine SDS-PAGE, comprise a fractionation profile as
depicted in FIG. 3, in which the proteins have carboxylesterase
activity.
[0019] Also included in the present invention is a formulation of
flea carboxylesterase proteins, in which the proteins, when
submitted to IEF-PAGE, comprise a fractionation profile as depicted
in FIG. 4, lane 3, lane 4, lane 5, lane 6 and/or lane 7, wherein
the proteins have carboxylesterase activity.
[0020] Another embodiment of the present invention is an isolated
flea protein or a formulation of flea proteins that hydrolyzes
.alpha.-napthyl acetate to produce .alpha.-napthol, when the
protein is incubated in the presence of .alpha.-napthyl acetate
contained in 20 mM Tris at pH 8.0 for about 15 minutes at about
37.degree. C.
[0021] Yet another embodiment of the present invention is an
isolated flea protein or a formulation of flea proteins that
hydrolyzes the methyl ester group of juvenile hormone to produce a
juvenile hormone acid.
[0022] Another embodiment of the present invention is a method to
identify a compound capable of inhibiting flea carboxylesterase
activity, the method comprising: (a) contacting an isolated flea
carboxylesterase with a putative inhibitory compound under
conditions in which, in the absence of the compound, the protein
has carboxylesterase activity; and (b) determining if the putative
inhibitory compound inhibits the activity. Also included in the
present invention is a test kit to identify a compound capable of
inhibiting flea carboxylesterase activity, the test kit comprising
an isolated flea carboxylesterase protein having esterase activity
and a means for determining the extent of inhibition of the
activity in the presence of a putative inhibitory compound.
[0023] Yet another embodiment of the present invention is a
therapeutic composition that is capable of reducing hematophagous
ectoparasite infestation. Such a therapeutic composition includes
at least one of the following protective compounds: an isolated
hematophagous ectoparasite carboxylesterase protein or a mimetope
thereof, an isolated carboxylesterase nucleic acid molecule that
hybridizes under stringent hybridization conditions with a
Ctenocephalides felis carboxylesterase gene, an isolated antibody
that selectively binds to a hematophagous ectoparasite
carboxylesterase protein, and an inhibitor of carboxylesterase
activity identified by its ability to inhibit the activity of a
flea carboxylesterase. A therapeutic composition of the present
invention can also include an excipient, an adjuvant and/or a
carrier. Preferred esterase nucleic acid molecule compounds of the
present invention include naked nucleic acid vaccines, recombinant
virus vaccines and recombinant cell vaccines. Also included in the
present invention is a method to protect an animal from
hematophagous ectoparasite infestation, comprising the step of
administering to the animal a therapeutic composition of the
present invention.
BRIEF DESCRIPTION OF THE FIGURES
[0024] FIG. 1 depicts SDS-PAGE analysis of DFP-labeled esterase
proteins.
[0025] FIG. 2 depicts carboxylesterase activity of certain esterase
proteins of the present invention.
[0026] FIG. 3 depicts SDS-PAGE analysis of carboxylesterase
activity of certain esterase proteins of the present invention.
[0027] FIG. 4 depicts IEF analysis of certain esterase proteins of
the present invention.
[0028] FIG. 5 depicts juvenile hormone esterase activity of certain
esterase proteins of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The present invention provides for isolated arthropod
esterase proteins, isolated arthropod esterase nucleic acid
molecules, antibodies directed against arthropod esterase proteins
and other inhibitors of arthropod esterase activity. As used
herein, the terms isolated arthropod esterase proteins and isolated
arthropod esterase nucleic acid molecules refers to esterase
proteins and esterase nucleic acid molecules derived from
arthropods and, as such, 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, nucleic acid molecules,
antibodies and inhibitors as therapeutic compositions to protect
animals from hematophagous ectoparasite infestation as well as in
other applications, such as those disclosed below.
[0030] Arthropod esterase proteins and nucleic acid molecules of
the present invention have utility because they represent novel
targets for anti-arthropod vaccines and drugs.
[0031] The products and processes of the present invention are
advantageous because they enable the inhibition of arthropod
development, metamorphosis, feeding, digestion and reproduction
processes that involve esterases. While not being bound by theory,
it is believed that expression of arthropod esterase proteins are
developmentally regulated, thereby suggesting that esterase
proteins are involved in arthropod development and/or reproduction.
The present invention is particularly advantageous because the
proteins of the present invention were identified in larval fleas,
thereby suggesting the importance of the proteins as developmental
proteins.
[0032] One embodiment of the present invention is an esterase
formulation that includes one or more esterase proteins capable of
binding to diisopropylfluorophosphate (DFP). A preferred embodiment
of an esterase formulation of the present invention comprises one
or more arthropod esterase proteins that range in molecular weight
from about 20 kilodaltons (kD) to about 200 kD, more preferably
from about 40 kD to about 100 kD, and even more preferably from
about 60 kD to about 75 kD, as determined by SDS-PAGE (sodium
dodecyl sulfate polyacrylamide gel electrophoresis). An even more
preferred formulation includes one or more flea esterase proteins
having elution (or migration) patterns as shown in FIG. 1.
[0033] Another embodiment of the present invention is a formulation
comprising one or more hematophagous ectoparasite carboxylesterase
(CE) proteins. The present invention includes the discovery that
such a formulation has general CE activity. General CE activity can
be identified using methods known to those of skill in the art and
described in the Examples section herein. A suitable formulation of
the present invention comprises one or more flea proteins capable
of hydrolyzing .alpha.-napthyl acetate to produce .alpha.-napthol
when the proteins are incubated in the presence of .alpha.-napthyl
acetate contained in 20 mm Tris at pH 8.0 for about 15 minutes at
about 37.degree. C. General CE activity can be identified following
such incubation by detecting the production of from about 0.3 to
about 2.5 absorbance units in the presence of Fast Blue when
measured at 590 nm.
[0034] A preferred CE formulation of the present invention includes
one or more flea CE proteins having acidic to neutral isoelectric
points, or pI values. An isoelectric pH, or pI, value refers to the
pH value at which a molecule has no net electric charge and fails
to move in an electric field. A preferred formulation of the
present invention includes one or more proteins having a pI value
ranging from about pI 2 to about 10, more preferably from about pI
3 to about 8, and even more preferably from about pI 4.7 to about
5.2, as determined by IEF-PAGE.
[0035] An esterase formulation, including a CE formulation, of the
present invention can be prepared by a method that includes the
steps of: (a) preparing an extract by isolating flea tissue,
homogenizing the tissue by sonication and clarifying the extract by
centrifugation at a low speed spin, e.g., about 18,000 rpm for
about 30 minutes; (b) recovering soluble proteins from said
centrifuged extract and applying the proteins to a
p-aminobenzamidine agarose bead column; (c) recovering unbound
protein from the column and clarifying by filtration; (d) applying
the clarified protein to a gel filtration column and eluting and
collecting fractions with esterase activity; (e) dilayzing the
eluate against 20 mM MES buffer, pH 6.0, containing 10 mM NaCl; (f)
applying the dialysate to a cation exchange chromatography column,
eluting protein bound to the column with a linear gradier of from
about 10 mM NaCl to about 1 M NaCl in 20 mM MES buffer, pH 6, and
collecting fractions having esterase activity; (g) adjusting the pH
of the resulting fractions to pH 7 and applying the fractions to an
anion exchange chromatography column; (h) eluting protein bound to
the column with a linear gradient of from about 0 to about 1 M NaCl
in 25 mM Tris buffer, pH 6.8 and collecting fractions having
esterase activity, such activity elutes from the column at about
170 mM NaCl.
[0036] Tissue can be obtained from unfed fleas or from fleas that
recently consumed a blood meal (i.e., blood-fed fleas). Such flea
tissues are referred to herein as, respectively, unfed flea and fed
flea tissue. Preferred flea tissue from which to obtain an esterase
formulation of the present invention includes pre-pupal larval
tissue, wandering flea larvae, 3rd instar tissue, fed adult tissue
and unfed adult tissue.
[0037] In a preferred embodiment, a CE formulation of the present
invention comprises a flea protein comprising amino acid sequence
SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:39, SEQ ID NO:40, SEQ ID
NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44 and/or SEQ ID
NO:53.
[0038] Another embodiment of the present invention is a juvenile
hormone esterase (JHE) formulation comprising one or more arthropod
JHE proteins, the arthropod being of the order Hemiptera, Anoplura,
Mallophaga, Diptera, Siphonaptera, Parasitiformes, Acariformes and
Acarina. The present invention includes the discovery that such a
formulation has JHE activity. JHE activity can be identified using
methods known to those of skill in the art and described in the
Examples section herein. A suitable formulation of the present
invention comprises one or more arthropod proteins capable of
hydrolyzing a methyl ester group of juvenile hormone to produce a
juvenile hormone acid. Preferably, such a protein is capable of
releasing of at least about 120 counts per minute when such a
protein is incubated in the presence of .sup.3H-juvenile hormone to
create a reaction mixture, the reaction mixture is combined with
isooctane, the aqueous phase is recovered and the amount of
.sup.3H-juvenile hormone present in that phase is determined. Such
a protein is also preferably capable of causing release of methane
thiol when such protein is incubated in the presence of methyl
1-heptylthioacetothioate (HEPTAT) using the method generally
disclosed in McCutchen et al., Insect Biochem. Molec. Biol., Vol.
25, No. 1, pg 119-126, 1995, which is incorporated in its entirety
by this reference.
[0039] In one embodiment, a juvenile hormone esterase formulation
of the present invention comprises a protein comprising amino acid
sequence SEQ ID NO:74.
[0040] According to the present invention, an arthropod that is not
of the order lepidoptera includes an arthropod of the order
Hemiptera, Anoplura, Mallophaga, Diptera, Siphonaptera,
Parasitiformes, Acariformes and Acarina. Preferred arthropods
include Hemiptera cimicidae, Hemiptera reduviidae, Anoplura
pediculidae, Anoplura pthiridae, Diptera culicidae, Diptera
simuliidae, Diptera psychodidae, Diptera ceratopogonidae, Diptera
chaoboridae, Diptera tabanidae, Diptera rhagionidae, athericidae,
Diptera chloropidae, Diptera muscidae, Diptera hippoboscidae,
Diptera calliphoridae, Diptera sarcophagidae, Diptera oestridae,
Diptera gastrophilidae, Diptera cuterebridae, Siphonaptera
ceratophyllidae, Siphonaptera leptopsyllidae, Siphonaptera
pulicidae, Siphonaptera tungidae, Parasitiformes dermanyssidae,
Acariformes tetranychidae, Acariformes cheyletide, Acariformes
demodicidae, Acariformes erthraeidae, Acariformes trombiculidae,
Acariformes psoroptidae, Acariformes sarcoptidae, Acarina argasidae
and Acarina ixodidae. Preferred Diptera muscidae include Musca,
Hydrotaea, Stomoxys Haematobia. Preferred Siphonaptera include
Ceratophyllidae nosopsyllus, Ceratophyllidae diamanus,
Ceratophyllidae ceratophyllus, Leptopsyllidae leptopsylla,
Pulicidae pulex, Pulicidae ctenocephalides, Pulicidae xenopsylla,
Pulicidae echidnophaga and Tungidae tunga. Preferred Parasitiformes
dermanyssidae include Ornithonyssus and Liponyssoides. Preferred
Acarina include Argasidae argas, Argasidae ornithodoros, Argasidae
otobius Ixodidae ixodes, Ixodidae hyalomma, Ixodidae nosomma,
Ixodidae rhipicephalus, Ixodidae boophilus, Ixodidae dermacentor;
Ixodidae haemaphysalus, Ixodidae amblyomma and Ixodidae
anocentor.
[0041] One embodiment of a JHE formulation of the present invention
is one or more arthropod JHE proteins that range in molecular
weight from about 20 kD to about 200 kD, more preferably from about
40 kD to about 100 kD, and even more preferably from about 60 kD to
about 75 kD, as determined by SDS-PAGE (sodium dodecyl sulfate
polyacrylamide gel electrophoresis).
[0042] A JHE formulation of the present invention can be prepared
by a method (hat includes the steps of: (a) preparing soluble
proteins from arthropod extracts as described above for CE
purification and purifying such soluble proteins by gel filtration;
(b) collecting fractions having JHE activity from the gel
filtration step, loading the fractions onto a cation exchange
column, eluting the cation exchange column with a linear gradient
of from about 10 mM NaCl to about 1 M NaCl in 20 mM MES buffer, pH
6 and collecting fractions having JHE activity; (c) adjusting the
pH of the collected fractions to about pH 7 are dialyzed against
about 10 mM phosphate buffer (pH 7.2) containing about 10 mM NaCl;
(d) applying the dialysate to a hydroxyapatite column, eluting
protein bound to the column with a linear gradient of from about 10
mM phosphate buffer (pH 7.2) containing 10 mM NaCl to about 0.5 M
phosphate buffer (pH 6.5) containing 10 mM NaCl and collecting
fractions having JHE activity; (e) dialyzing the fractions against
20 mM Tris buffer (pH 8.0) containing 10 mM NaCl; (f) applying the
dialysate an anion exchange chromatography column and eluting
protein bound to the column with a linear gradient of from about 10
mM to about 1 M NaCl in 20 mM Tris buffer, pH 8 and collecting
fractions containing JHE activity.
[0043] A JHE formulation of the present invention can be prepared
by a method that includes the steps of: (a) preparing flea extracts
as described herein in the Examples section and applying the
extract to p-aminobenzamidine linked agarose beads and collecting
protein not bound to the beads; (b) applying the unbound protein to
a Superdex 200 HR gel filtration column and collecting fractions
having JHE activity; (c) applying the fractions to an anion
exchange chromatography column, eluting the anion exchange column
with a linear gradient of 0 to 1 M NaCl in 25 mM Tris buffer, pH
6.8 and collecting fractions having JHE activity; (d) dialyzing the
fractions overnight against about 1 L of 20 mM Tris buffer, pH 8.0,
containing 10 mM NaCl; (e) applying the dialysate to a Poros 10 HQ
anion exchange column, eluting the column with buffer containing
about 120 mM NaCl and collecting fractions having JHE activity.
[0044] Suitable arthropods from which to isolate a JHE formulation
of the present invention include, but are not limited to
agricultural pests, stored product pests, forest pests, structural
pests or animal health pests. Suitable agricultural pests of the
present invention include, but are not limited to Colorado potato
beetle., corn earworms, fleahoppers, weevils, pink boll worms,
cotton aphids, beet armyworms, lygus bugs, hessian flies, sod
webworms, whites grubs, diamond back moths, white flies,
planthoppers, leafhoppers, mealy bugs, mormon crickets and mole
crickets. Suitable stored product pests of the present invention
include, but are not limited to dermestids, anobeids, saw toothed
grain beetles, indian mealmoths, flour beetles, long-horn wood
boring beetles and metallic wood boring beetles. Suitable forest
pests of the present invention include, but are not limited to
southern pine bark bettles, gypsy moths, elm beetles, ambrosia
bettles, bag worms, tent worms and tussock moths. Suitable
structural pests of the present invention include, but are not
limited to, bess beetles, termites, fire ants, carpenter ants,
wasps, hornets, cockroaches, silverfish, Musca domestica and Musca
autumnalis. Suitable animal health pests of the present invention
include, but are not limited to fleas, ticks, mosquitoes, black
flies, lice, true bugs, sand flies, Psychodidae, tsetse flies,
sheep blow flies, cattle grub, mites, horn flies, heel flies, deer
flies, Culicoides and warble flies. Preferred arthropods from which
to isolate a JHE formulation of the present invention include
fleas, midges, mosquitos, sand flies, black flies, horse flies,
snipe flies, louse flies, horn flies, deer flies, tsetse flies,
buffalo flies, blow flies, stable flies, myiasis-causing flies,
biting gnats, lice, mites, bee, wasps, ants, true bugs and ticks,
preferably fleas, ticks and blow flies, and more preferably fleas.
Preferred fleas from which to isolate JHE proteins include
Ctenocephalides, Ceratophyllus, Diamanus, Echidnophaga,
Nosopsyllus, Pulex, Tunga, Oropsylla, Orchopeus and Xenopsylla.
More preferred fleas include Ctenocephalides felis, Ctenocephalides
canis, Ceratophyllus pulicidae, Pulex irritans, Oropsylla
(Thrassis) bacchi, Oropsylla (Diamanus) montana, Orchopeus howardi,
Xenopsylla cheopis and Pulex simulans, with C. felis being even
more preferred.
[0045] Suitable tissue from which to isolate a JHE formulation of
the present invention includes unfed fleas or fleas that recently
consumed a blood meal (i.e., blood-fed fleas). Such flea tissues
are referred to herein as, respectively, unfed flea and fed flea
tissue. Preferred flea tissue from which to obtain a JHE
formulation of the present invention includes pre-pupal larval
tissue, 3rd instar tissue, fed or unfed adult tissue, with unfed
adult gut tissue being more preferred than fed or unfed whole adult
tissue. It is of note that a JHE formulation of the present
invention obtained from pre-pupal larval tissue does not hydrolyze
.alpha.-napthyl acetate.
[0046] Another embodiment of the present invention is an esterase
formulation comprising a combination of one or more arthropod CE
and JHE proteins of the present invention. Suitable arthropods from
which to isolate a combined CE and JHE formulation include those
arthropods described herein for the isolation of a JHE formulation
of the present invention. Preferred arthropods from which to
isolate a combined CE and JHE formulation include fleas, midges,
mosquitos, sand flies, black flies, horse flies, horn flies, deer
flies, tsetse flies, buffalo flies, blow flies, stable flies,
myiasis-causing flies, biting gnats, lice, bee, wasps, ants, true
bugs and ticks, preferably fleas, ticks and blow flies, and more
preferably fleas. Suitable flea tissue from which to isolate a
combined CE and JHE formulation of the present invention includes
3rd instar tissue, fed or unfed adult tissue and unfed adult
tissue, with unfed adult gut tissue being more preferred than fed
or unfed whole adult tissue.
[0047] In one embodiment, a formulation of the present invention
comprises an esterase having both CE and JHE activity. Preferably,
a formulation of the present invention that comprises an esterase
having both CE and JHE activity comprises a flea protein comprising
amino acid sequence SEQ ID NO:8 and/or SEQ ID NO:37.
[0048] Another embodiment of the present invention is an isolated
protein comprising an arthropod esterase 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 refers to one or more proteins or at
least one protein. 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. Furthermore, a compound "selected from
the group consisting of" refers to one or more of the compounds in
the list that follows, including mixtures (i.e., combinations) of
two or more of the compounds. 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.
[0049] As used herein, an isolated arthropod esterase protein can
be a full-length protein or any homolog of such a protein. An
isolated protein of the present invention, including a homolog, can
be identified in a straight-forward manner by the protein's ability
to elicit an immune response against arthropod esterase proteins,
to hydrolyze .alpha.-napthyl acetate, to hydrolyze the methyl ester
group of juvenile hormone or bind to DFP. Esterase proteins of the
present invention include CE and JHE proteins. As such, an esterase
protein of the present invention can comprise a protein capable of
hydrolyzing .alpha.-napthyl acetate, hydrolyzing the methyl ester
group of juvenile hormone and/or binding to DFP. Examples of
esterase homologs include esterase 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 homolog
includes at least one epitope capable of eliciting an immune
response against an arthropod esterase protein. That is, when the
homolog 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 arthropod esterase protein. The ability of a protein to
effect an immune response, can be measured using techniques known
to those skilled in the art. Esterase protein homologs of the
present invention also include esterase proteins that hydrolyze
.alpha.-napthyl acetate and/or that hydrolyze the methyl ester
group of juvenile hormone.
[0050] Arthropod esterase protein homologs can be the result of
natural allelic variation or natural mutation. Esterase protein
homologs 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 nucleic acid
techniques to effect random or targeted mutagenesis.
[0051] Isolated esterase proteins of the present invention have the
further characteristic of being encoded by nucleic acid molecules
that hybridize under stringent hybridization conditions to a gene
encoding a Ctenocephalides felis protein (i.e., a C. felis esterase
gene). As used herein, stringent hybridization conditions refer to
standard hybridization conditions under which nucleic acid
molecules, including oligonucleotides, are used to identify similar
nucleic acid molecules. Such standard conditions are disclosed, for
example, in Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Labs Press, 1989; Sambrook et al.,
ibid., is incorporated by reference herein in its entirety.
Stringent hybridization conditions typically permit isolation of
nucleic acid molecules having at least about 70% nucleic acid
sequence identity with the nucleic acid molecule being used to
probe in the hybridization reaction. Formulae to calculate the
appropriate hybridization and wash conditions to achieve
hybridization permitting 30% or less mismatch of nucleotides are
disclosed, for example, in Meinkoth et al., 1984, Anal. Biochem.
138, 267-284; Meinkoth et al., ibid., is incorporated by reference
herein in its entirety.
[0052] As used herein, a C. felis esterase gene includes all
nucleic acid sequences related to a natural C. felis esterase gene
such as regulatory regions that control production of the C. felis
esterase protein encoded by that gene (such as, but not limited to,
transcription, translation or post-translation control regions) as
well as the coding region itself. In one embodiment, a C. felis
esterase gene of the present invention includes the 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:17, SEQ ID NO:18, SEQ ID NO:20, SEQ
ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26,
SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID
NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ
ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59,
SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID
NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic
acid molecule encoding a protein comprising amino acid sequence SEQ
ID NO:74. Nucleic acid sequence SEQ ID NO:1 represents the deduced
sequence of the coding strand of a PCR amplified nucleic acid
molecule denoted herein as nfE1.sub.401, the production of which is
disclosed in the Examples. The complement of SEQ ID NO:1
(represented herein by SEQ ID NO:3) refers to the nucleic acid
sequence of the strand 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 complementary to (i.e.,
can form a complete double helix with) the strand for which the
sequence is cited.
[0053] Nucleic acid sequence SEQ ID NO:4 represents the deduced
sequence of the coding strand of a PCR amplified nucleic acid
molecule denoted herein as nfE2.sub.364, the production of which is
disclosed in the Examples. The complement of SEQ ID NO:4 is
represented herein by SEQ ID NO:6.
[0054] Nucleic acid sequence SEQ ID NO:7 represents the deduced
sequence of the coding strand of a PCR amplified nucleic acid
molecule denoted herein as nfE3.sub.421, the production of which is
disclosed in the Examples. The complement of SEQ ID NO:7 is
represented herein by SEQ ID NO:9.
[0055] Nucleic acid sequence SEQ ID NO:10 represents the deduced
sequence of the coding strand of a PCR amplified nucleic acid
molecule denoted herein as nfE4.sub.524, the production of which is
disclosed in the Examples. The complement of SEQ ID NO:10 is
represented herein by SEQ ID NO:12. Nucleic acid sequence SEQ ID
NO:13 represents the deduced sequence of the coding strand of an
apparent coding region of a complementary DNA (cDNA) nucleic acid
molecule denoted herein as nfE5.sub.1982, the production of which
is disclosed in the Examples. The complement of SEQ ID NO:13 is
represented herein by SEQ ID NO:15.
[0056] Nucleic acid sequence SEQ ID NO:18 represents the deduced
sequence of the coding strand of an apparent coding region of a
cDNA nucleic acid molecule denoted herein as nfE6.sub.1792, the
production of which is disclosed in the Examples. The complement of
SEQ ID NO:18 is represented herein by SEQ ID NO:20.
[0057] Nucleic acid sequence SEQ ID NO:24 represents the deduced
sequence of the coding strand of an apparent coding region of a
cDNA nucleic acid molecule denoted herein as nfE7.sub.2936, the
production of which is disclosed in the Examples. The complement of
SEQ ID NO:24 is represented herein by SEQ ID NO:26.
[0058] Nucleic acid sequence SEQ ID NO:30 represents the deduced
sequence of the coding strand of an apparent coding region of a
cDNA nucleic acid molecule denoted herein as nfE8.sub.2801, the
production of which is disclosed in the Examples. The complement of
SEQ ID NO:30 is represented herein by SEQ ID NO:32.
[0059] Nucleic acid sequence SEQ ID NO:36 represents the deduced
sequence of the coding strand of an apparent coding region of a
cDNA nucleic acid molecule denoted herein as nfE9.sub.2007, the
production of which is disclosed in the Examples. The complement of
SEQ ID NO:36 is represented herein by SEQ ID NO:38.
[0060] Nucleic acid sequence SEQ ID NO:57 represents the deduced
sequence of the coding strand of an apparent coding region of a
cDNA nucleic acid molecule denoted herein as nfE5.sub.2144, the
production of which is disclosed in the Examples. The complement of
SEQ ID NO:57 is represented herein by SEQ ID NO:59.
[0061] Nucleic acid sequence SEQ ID NO:67 represents the deduced
sequence of the coding strand of an apparent coding region of a
cDNA nucleic acid molecule denoted herein as nfE10.sub.1987, the
production of which is disclosed in the Examples. The complement of
SEQ ID NO:67 is represented herein by SEQ ID NO:69.
[0062] It should be noted that since nucleic acid sequencing
technology is not entirely error-free, the nucleic acid sequences
and amino acid sequences presented herein represent, respectively,
apparent nucleic acid sequences of nucleic acid molecules of the
present invention and apparent amino acid sequences of esterase
proteins of the present invention.
[0063] In another embodiment, a C. felis esterase gene 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:17, SEQ ID NO:18, SEQ ID NO:20, SEQ
ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26,
SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID
NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ
ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59,
SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID
NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic
acid molecule encoding a protein comprising amino acid sequence SEQ
ID NO:74. An allelic variant of a C. felis esterase gene is a gene
that occurs at essentially the same locus (or loci) in the genome
as the gene including 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:17, SEQ ID NO:18, SEQ
ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24,
SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID
NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ
ID NO:36, SEQ ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57,
SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID
NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76
and/or a nucleic acid molecule encoding a protein comprising amino
acid sequence SEQ ID NO:74, but which, due to natural variations
caused by, for example, mutation or recombination, has a similar
but not identical sequence. Allelic variants typically encode
proteins having similar activity to that of the protein encoded by
the gene to which they are being compared. Allelic variants can
also comprise alterations in the 5' or 3' untranslated regions of
the gene (e.g., in regulatory control regions). Allelic variants
are well known to those skilled in the art and would be expected to
be found within a given arthropod since the genome is diploid
and/or among a group of two or more arthropods.
[0064] The minimal size of an esterase protein homolog of the
present invention is a size sufficient to be encoded by a nucleic
acid molecule capable of foiling a stable hybrid (i.e., hybridize
under stringent hybridization conditions) with the complementary
sequence of a nucleic acid molecule encoding the corresponding
natural protein. As such, the size of the nucleic acid molecule
encoding such a protein homolog is dependent on nucleic acid
composition and percent homology between the nucleic acid molecule
and complementary sequence. It should also be noted that the extent
of homology required to form a stable hybrid can vary depending on
whether the homologous sequences are interspersed throughout the
nucleic acid molecules or are clustered (i.e., localized) in
distinct regions on the nucleic acid molecules. The minimal size of
such nucleic acid molecules is typically at least about 12 to about
15 nucleotides in length if the nucleic acid molecules are GC-rich
and at least about 15 to about 17 bases in length if they are
AT-rich. As such, the minimal size of a nucleic acid molecule used
to encode an esterase protein homolog of the present invention is
from about 12 to about 18 nucleotides in length. Thus, the minimal
size of an esterase protein homolog 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 such a nucleic
acid molecule in that the nucleic acid molecule can include a
portion of a gene, an entire gene, multiple genes, or portions
thereof. 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.
[0065] One embodiment of the present invention includes an
arthropod esterase protein having CE enzyme activity. Such a CE
protein preferably includes: a catalytic triad of
serine--lhistidine--glutamic acid as well as the essential amino
acids arginine and aspartic acid at positions similar to those
described for juvenile hormone esterase, for example by Ward et
al., 1992, Int J Biochem 24: 1933-1941; this reference is
incorporated by reference herein in its entirety. Analysis of the
apparent full-length protein sequences disclosed herein indicates
that each of these amino acid sequences includes these amino acid
motifs, as well as surrounding consensus sequences.
[0066] Suitable arthropods from which to isolate esterase proteins
having general CE activity of the present invention (including
isolation of the natural protein or production of the protein by
recombinant or synthetic techniques) preferably include insects and
acarines but not Culicidae, Drosophilidae, Calliphoridae,
Sphingidae, Lymantriidae, Noctuidae, Fulgoroidae and Aphididae.
Preferred arthropods from which to isolate CE proteins having
general CE activity include fleas, ticks, black flies, lice, true
bugs, sand flies, Psychodidae, tsetse flies, cattle grub, mites,
horn flies, heel flies, deer flies, Culicoides and warble flies.
Preferred arthropods from which to isolate an esterase proteins
having general CE activity include fleas, midges, sand flies, black
flies, horse flies, snipe flies, louse flies, hlo'n flies, deer
flies, tsetse flies, buffalo flies, blow flies, stable flies,
myiasis-causing flies, biting gnats, lice, mites, bee, wasps, ants,
true bugs and ticks, preferably fleas, ticks and blow flies, and
more preferably fleas. Preferred fleas from which to isolate
esterase proteins having general CE activity include
Ctenocephalides, Ceratophyllus, Diamanus, Echidnophaga,
Nosopsyllus, Pulex, Tunga, Oropsylla, Orchopeus and Xenopsylla.
More preferred fleas include Ctenocephalides felis, Ctenocephalides
canis, Ceratophyllus pulicidae, Pulex irritans, Oropsylla(Thrassis)
bacchi, Oropsylla (Diamanus) montana, Orchopeus howardi, Xenopsylla
cheopis and Pulex simulans, with C. felis being even more
preferred.
[0067] A preferred arthropod esterase protein of the present
invention is a compound that when administered to an animal in an
effective manner, is capable of protecting that animal from
hematophagous ectoparasite infestation. In accordance with the
present invention, the ability of an esterase protein of the
present invention to protect an animal from hematophagous
ectoparasite infestation refers to the ability of that protein to,
for example, treat, ameliorate and/or prevent infestation caused by
hematophagous arthropods. In particular, the phrase "to protect an
animal from hematophagous ectoparasite infestation" refers to
reducing the potential for hematophagous ectoparasite population
expansion on and around the animal (i.e., reducing the
hematophagous ectoparasite burden). Preferably, the hematophagous
ectoparasite population size is decreased, optimally to an extent
that the animal is no longer bothered by hematophagous
ectoparasites. A host animal, as used herein, is an animal from
which hematophagous ectoparasites can feed by attaching to and
feeding through the skin of the animal. Hematophagous
ectoparasites, 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 hematophagous ectoparasite 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 hematophagous
ectoparasites, but also hematophagous ectoparasite eggs and/or
hematophagous ectoparasite larvae. The environment can be of any
size such that hematophagous ectoparasites 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 hematophagous ectoparasites infest an animal. As such, it is
desirable not only to reduce the hematophagous ectoparasite burden
on an animal per se, but also to reduce the hematophagous
ectoparasite burden in the environment of the animal. In one
embodiment, an esterase protein of the present invention can elicit
an immune response (including a humoral and/or cellular immune
response) against a hematophagous ectoparasite.
[0068] Suitable hematophagous ectoparasites to target include any
hematophagous ectoparasite that is essentially incapable of
infesting an animal administered an esterase protein of the present
invention. As such, a hematophagous ectoparasite to target includes
any hematophagous ectoparasite that produces a protein having one
or more epitopes that can be targeted by a humoral and/or cellular
immune response against an esterase protein of the present
invention and/or that can be targeted by a compound that otherwise
inhibits esterase activity (e.g., a compound that inhibits
hydrolysis of .alpha.-napthyl acetate, hydrolysis of the methyl
ester group of juvenile hormone, and/or binds to DFP), thereby
resulting in the decreased ability of the hematophagous
ectoparasite to infest an animal. Preferred hematophagous
ectoparasite to target include ectoparasites disclosed herein as
being useful in the production of esterase proteins of the present
invention.
[0069] The present invention also includes mimetopes of esterase
proteins of the present invention. As used herein, a mimetope of an
esterase protein of the present invention refers to any compound
that is able to mimic, the activity of such a protein (e.g.,
ability to elicit an immune response against an arthropod esterase
protein of the present invention and/or ability to inhibit esterase
activity), often because the mimetope has a structure that mimics
the esterase protein. It is to be noted, however, that the mimetope
need not have a structure similar to an esterase protein as long as
the mimetope functionally mimics the protein. Mimetopes can be, but
are not limited to: peptides that have been modified to decrease
their susceptibility to degradation; anti-idiotypic and/or
catalytic antibodies, or fragments thereof; non-proteinaceous
immunogenic portions of an isolated protein (e.g., carbohydrate
signatures); synthetic or natural organic or inorganic molecules,
including nucleic acids; and/or any other peptidomimetic
compounds.
[0070] Mimetopes of the present invention can be designed using
computer-generated structures of esterase 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,
(e.g., an esterase substrate, an esterase substrate analog, or an
anti-esterase antibody). A preferred mimetope is a peptidomimetic
compound that is structurally and/or functionally similar to an
esterase protein of the present invention, particularly to the
active site of the esterase protein.
[0071] The present invention also includes mimetopes of esterase
proteins of the present invention. As used herein, a mimetope of an
esterase protein of the present invention refers to any compound
that is able to mimic the activity of such an esterase protein,
often because the mimetope has a structure that mimics the esterase
protein. Mimetopes can be, but are not limited to: peptides that
have been modified to decrease their susceptibility to degradation;
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.
[0072] One embodiment of an arthropod esterase protein of the
present invention is a fusion protein that includes an arthropod
esterase 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 an esterase protein; and/or
assist purification of an esterase 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 esterase-containing
domain of the protein and can be susceptible to cleavage in order
to enable straight-forward recovery of an esterase 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 esterase-containing
domain. Preferred fusion segments include a metal binding domain
(e.g., a polyhistidine 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, 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. Examples of particularly preferred fusion proteins
of the present invention include PHIS-PfE6.sub.540,
PHIS-PfE7.sub.275, PHIS-PfE7.sub.570, PHIS-PfE8.sub.570 and
PHIS-PfE9.sub.528, production of which are disclosed herein.
[0073] In another embodiment, an arthropod esterase protein of the
present invention also includes at least one additional protein
segment that is capable of protecting an animal from hematophagous
ectoparasite infestations. Such a multivalent protective protein
can be produced by culturing a cell transformed with a nucleic acid
molecule comprising two or more nucleic acid domains joined
together in such a manner that the resulting nucleic acid molecule
is expressed as a multivalent protective compound containing at
least two protective compounds, or portions thereof, capable of
protecting an animal from hematophagous ectoparasite infestation
by, for example, targeting two different arthropod proteins.
[0074] Examples of multivalent protective compounds include, but
are not limited to, an esterase protein of the present invention
attached to one or more compounds protective against one or more
arthropod compounds. Preferred second compounds are proteinaceous
compounds that effect active immunization (e.g., antigen vaccines),
passive immunization (e.g., antibodies), or that otherwise inhibit
a arthropod activity that when inhibited can reduce hematophagous
ectoparasite burden on and around an animal. Examples of second
compounds include a compound that inhibits binding between an
arthropod protein and its ligand (e.g., a compound that inhibits
flea ATPase activity or a compound that inhibits binding of a
peptide or steroid hormone to its receptor), a compound that
inhibits hormone (including peptide or steroid hormone) synthesis,
a compound that inhibits vitellogenesis (including production of
vitellin and/or transport and maturation thereof into a major egg
yolk protein), a compound that inhibits fat body function, a
compound that inhibits muscle action, a compound that inhibits the
nervous system, a compound that inhibits the immune system and/or a
compound that inhibits hematophagous ectoparasite feeding. Examples
of second compounds also include proteins obtained from different
stages of hematophagous ectoparasite development. Particular
examples of second compounds include, but are not limited to,
serine proteases, cysteine proteases, aminopeptidases, serine
protease inhibitor proteins, calreticulins, larval serum proteins
and echdysone receptors, as well as antibodies to and inhibitors of
such proteins. In one embodiment, an arthropod esterase protein of
the present invention is attached to one or more additional
compounds protective against hematophagous ectoparasite
infestation. In another embodiment, one or more protective
compounds, such as those listed above, can be included in a
multivalent vaccine comprising an arthropod esterase protein of the
present invention and one or more other protective molecules as
separate compounds.
[0075] A preferred isolated protein of the present invention is a
protein encoded by a nucleic acid molecule that hybridizes under
stringent hybridization conditions with nucleic acid molecules
nfE1.sub.401, nfE2.sub.364, nfE3.sub.421, nfE4.sub.524,
nfE5.sub.1982, nfE5.sub.1515, nfE5.sub.2144, nfE5.sub.1650,
nfE6.sub.1488, nfE6.sub.1792, nfE6.sub.1650, nfE7.sub.2836,
nfE7.sub.1788, nfE7.sub.1710, nfE7.sub.650, nfE8.sub.2801,
nfE8.sub.1785, nfE8.sub.1710, nfE9.sub.2007, nfE9.sub.1584,
nfE9.sub.1540, nfE10.sub.1987 and/or nfE10.sub.1590. A further
preferred isolated protein is encoded by a nucleic acid molecule
that hybridizes under stringent hybridization conditions with a
nucleic acid molecule having nucleic acid sequence SEQ ID NO:3, SEQ
ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ TD NO:17, SEQ
ID NO:20, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32,
SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:52, SEQ ID NO:59, SEQ ID
NO:61, SEQ ID NO:69 and/or SEQ ID NO:71.
[0076] Translation of SEQ ID NO:1 suggests that nucleic acid
molecule nfE1.sub.401 encodes a non-full-length arthropod esterase
protein of about 103 amino acids, referred to herein as
PfE1.sub.103, represented by SEQ ID NO:2, assuming the first codon
spans from nucleotide 92 through nucleotide 94 of SEQ ID NO:1.
[0077] Comparison of amino acid sequence SEQ ID NO:2 (i.e., the
amino acid sequence of PfE1.sub.103) with amino acid sequences
reported in GenBank indicates that SEQ ID NO:2, showed the most
homology, i.e., about 33% identity, between SEQ ID NO:2 and alpha
esterase protein from Drosophila melanogaster.
[0078] Translation of SEQ ID NO:4 suggests that nucleic acid
molecule nfE2.sub.364 encodes a non-full-length arthropod esterase
protein of about 121 amino acids, referred to herein as
PfE2.sub.121, represented by SEQ ID NO:5, assuming the first codon
spans from nucleotide 2 through nucleotide 4 of SEQ ID NO:4.
[0079] Comparison of amino acid sequence SEQ ID NO:5 (i.e., the
amino acid sequence of PfE2.sub.121) with amino acid sequences
reported in GenBank indicates that SEQ ID NO:5, showed the most
homology, i.e., about 38% identity, between SEQ ID NO:5 and alpha
esterase protein from Drosophila melanogaster.
[0080] Translation of SEQ ID NO:7 suggests that nucleic acid
molecule nfE3.sub.421, encodes a non-full-length arthropod esterase
protein of about 103 amino acids, referred to herein as
PfE3.sub.103, represented by SEQ ID NO:8, assuming the first codon
spans from nucleotide 113 through nucleotide 115 of SEQ ID
NO:7.
[0081] Comparison of amino acid sequence SEQ ID NO:8 (i.e., the
amino acid sequence of PfE3.sub.103) with amino acid sequences
reported in GenBank indicates that SEQ ID NO:5, showed the most
homology, i.e., about 39% identity, between SEQ ID NO:5 and alpha
esterase protein from Drosophila melanogaster.
[0082] Translation of SEQ ID NO:10 suggests that nucleic acid
molecule nfE4.sub.524 encodes a non-full-length arthropod esterase
protein of about 137 amino acids, referred to herein as
PfE4.sub.137, represented by SEQ ID NO:11, assuming the first codon
spans from nucleotide 113 through nucleotide 115 of SEQ ID
NO:10.
[0083] Comparison of amino acid sequence SEQ ID NO:11 (i.e., the
amino acid sequence of PfE4.sub.137) with amino acid sequences
reported in GenBank indicates that SEQ ID NO:11, showed the most
homology, i.e., about 30% identity, between SEQ ID NO:11 and
Leptinotarsa decemlineata acetylcholinesterase.
[0084] Translation of SEQ ID NO:57 suggests that nucleic acid
molecule nfE5.sub.2144 encodes a full-length arthropod esterase
protein of about 550 amino acids, referred to herein as
PfE5.sub.550, represented by SEQ ID NO:58, assuming an open reading
frame in which the initiation codon spans from nucleotide 30
through nucleotide 32 of SEQ ID NO:57 and the termination (stop)
codon spans from nucleotide 1680 through nucleotide 1682 of SEQ ID
NO:57. The complement of SEQ ID NO:57 is represented herein by SEQ
ID NO:59. The coding region encoding PfE5.sub.550 is represented by
the nucleic acid molecule nfE5.sub.1650, having a coding strand
with the nucleic acid sequence represented by SEQ ID NO:60 and a
complementary strand with nucleic acid sequence SEQ ID NO:61. The
deduced amino acid sequence of PfE5.sub.550 (i.e., SEQ ID NO:58)
predicts that PfE5.sub.550 has an estimated molecular weight of
about 61.8 kD and an estimated pI of about 5.5.
[0085] Comparison of amino acid sequence SEQ ID NO:58 (i.e., the
amino acid sequence of PfE5.sub.550) with amino acid sequences
reported in GenBank indicates that SEQ ID NO:58 showed the most
homology, i.e., about 36% identity between SEQ ID NO:58 and
Drosophila melanogaster alpha esterase protein.
[0086] Translation of SEQ ID NO:18 suggests that nucleic acid
molecule nfE6.sub.1792 encodes a full-length arthropod esterase
protein of about 550 amino acids, referred to herein as
PfE6.sub.550, represented by SEQ ID NO:19, assuming an open reading
frame having an initiation codon spanning from nucleotide 49
through nucleotide 51 of SEQ ID NO:18 and a stop codon spanning
from nucleotide 1699 through nucleotide 1701 of SEQ ID NO:18. The
coding region encoding PfE6.sub.550, is represented by nucleic acid
molecule nfE6.sub.1650, having a coding strand with the nucleic
acid sequence represented by SEQ ID NO:21 and a complementary
strand with nucleic acid sequence SEQ ID NO:22. The proposed mature
protein, denoted herein as PfE6.sub.530, contains about 530 amino
acids which is represented herein as SEQ ID NO:53. The nucleic acid
molecule encoding PfE6.sub.530 is denoted herein as nfE6.sub.1590
and has a coding strand having the nucleic acid sequence SEQ ID
NO:23. The deduced amino acid sequence SEQ ID NO:19 suggests a
protein having a molecular weight of about 61.8 kD and an estimated
pI of about 5.5.
[0087] Comparison of amino acid sequence SEQ ID NO:19 (i.e., the
amino acid sequence of PfE6.sub.550) with amino acid sequences
reported in GenBank indicates that SEQ ID NO:19 showed the most
homology, i.e., about 28% identity between SEQ ID NO:19 and
Drosophila melanogaster alpha esterase protein.
[0088] Translation of SEQ ID NO:24 suggests that nucleic acid
molecule nfE7.sub.283, encodes a full-length arthropod esterase
protein of about 596 amino acids, referred to herein as
PfE7.sub.596, represented by SEQ ID NO:25, assuming an open reading
frame having an initiation codon spanning from nucleotide 99
through nucleotide 101 of SEQ ID NO:24 and a stop codon spanning
from nucleotide 1887 through nucleotide 1889 of SEQ ID NO:24. The
coding region encoding PfE7.sub.596, is represented by nucleic acid
molecule nfE7.sub.1788, having a coding strand with the nucleic
acid sequence represented by SEQ ID NO:28 and a complementary
strand with nucleic acid sequence SEQ ID NO:29. The proposed mature
protein, denoted herein as PfE7.sub.570, contains about 570 amino
acids which is represented herein as SEQ ID NO:54. The nucleic acid
molecule encoding PfE7.sub.570 is denoted herein as nfE7.sub.1710
and has a coding strand having the nucleic acid sequence SEQ ID
NO:27. The deduced amino acid sequence SEQ ID NO:25 suggests a
protein having a molecular weight of about 68.7 kD and an estimated
pI of about 6.1.
[0089] Comparison of amino acid sequence SEQ ID NO:25 (i.e., the
amino acid sequence of PfE7.sub.596) with amino acid sequences
reported in GenBank indicates that SEQ ID NO:25 showed the most
homology, i.e., about 27% identity between SEQ ID NO:25 and
Drosophila melanogaster alpha esterase protein.
[0090] Translation of SEQ ID NO:30 suggests that nucleic acid
molecule nfE8.sub.2801, encodes a full-length arthropod esterase
protein of about 595 amino acids, referred to herein as
PfE8.sub.595, represented by SEQ ID NO:31, assuming an open reading
frame having an initiation codon spanning from nucleotide 99
through nucleotide 101 of SEQ ID NO:30 and a stop codon spanning
from nucleotide 1884 through nucleotide 1886 of SEQ ID NO:30. The
coding region encoding PfE8.sub.595, is represented by nucleic acid
molecule nfE8.sub.1785, having a coding strand with the nucleic
acid sequence represented by SEQ ID NO:34 and a complementary
strand with nucleic acid sequence SEQ ID NO:35. The proposed mature
protein, denoted herein as PfE8.sub.570, contains about 570 amino
acids which is represented herein as SEQ ID NO:55. The nucleic acid
molecule encoding PfE8.sub.570 is denoted herein as nfE8.sub.1710
and has a coding strand having the nucleic acid sequence SEQ ID
NO:33. The deduced amino acid sequence SEQ ID NO:31 suggests a
protein having a molecular weight of about 68.6 kD and an estimated
pI of about 6.1.
[0091] Comparison of amino acid sequence SEQ ID NO:31 (i.e., the
amino acid sequence of PfE8.sub.595) with amino acid sequences
reported in GenBank indicates that SEQ ID NO:31 showed the most
homology, i.e., about 28% identity between SEQ ID NO:31 and
estalpha-2 esterase of Culex pipiens quinquefasciatus.
[0092] Translation of SEQ ID NO:36 suggests that nucleic acid
molecule nfE9.sub.2007 encodes a full-length arthropod esterase
protein of about 528 amino acids, referred to herein as
PfE9.sub.528, represented by SEQ ID NO:37, assuming an open reading
frame having an initiation codon spanning from nucleotide 11
through nucleotide 13 of SEQ ID NO:36 and a stop codon spanning
from nucleotide 1595 through nucleotide 1597 of SEQ ID NO:36. The
coding region encoding PfE9.sub.528, is represented by nucleic acid
molecule nfE9.sub.1584, having a coding strand with the nucleic
acid sequence represented by SEQ ID NO:51 and a complementary
strand with nucleic acid sequence SEQ ID NO:52. The deduced amino
acid sequence SEQ ID NO:37 suggests a protein having a molecular
weight of about 60 kD and an estimated pI of about 5.43.
[0093] Comparison of amino acid sequence SEQ ID NO:37 (i.e., the
amino acid sequence of PfE9.sub.528) with amino acid sequences
reported in GenBank indicates that SEQ ID NO:37 showed the most
homology, i.e., about 37% identity between SEQ ID NO:37 and alpha
esterase protein from Drosophila melanogaster.
[0094] Translation of SEQ ID NO:67 suggests that nucleic acid
molecule nfE10.sub.1997 encodes a full-length flea esterase protein
of about 530 amino acids, referred to herein as PfE10.sub.530,
having amino acid sequence SEQ ID NO:68, assuming an open reading
frame in which the initiation codon spans from nucleotide 231
through nucleotide 233 of SEQ ID NO:67 and a stop codon spanning
from nucleotide 1821 through nucleotide 1823 of SEQ ID NO:67. The
complement of SEQ ID NO:67 is represented herein by SEQ ID NO:69.
The coding region encoding PfE10.sub.530, is represented by nucleic
acid molecule nfE10.sub.590, having a coding strand with the
nucleic acid sequence represented by SEQ ID NO:70 and a
complementary strand with nucleic acid sequence SEQ ID NO:71. The
amino acid sequence of PfE10.sub.530 (i.e., SEQ ID NO:68) predicts
that PfE10.sub.530 has an estimated molecular weight of about 59.5
kD and an estimated pI of about 5.5.
[0095] Comparison of amino acid sequence SEQ ID NO:68 (i.e., the
amino acid sequence of PfE10.sub.530) with amino acid sequences
reported in GenBank indicates that SEQ ID NO:68 showed the most
homology, i.e., about 30% identity between SEQ ID NO:68 and Culex
pipens esterase b1 precurser protein (swissprot #16854).
[0096] More preferred arthropod esterase proteins of the present
invention include proteins comprising amino acid sequences that are
at least about 40%, preferably at least about 45%, more preferably
at least about 50%, even more preferably at least about 55%, even
more preferably at least about 60%, even more preferably at least
about 70%, even more preferably at least about 80%, even more
preferably at least about 90%, and even more preferably at least
about 95%, identical to amino acid sequence SEQ ID NO:2, SEQ ID
NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID
NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ
ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53,
SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID
NO:73 and/or SEQ ID NO:74.
[0097] More preferred arthropod esterase proteins of the present
invention include proteins encoded by a nucleic acid molecule
comprising at least a portion of nfE1.sub.401, nfE2.sub.364,
nfE3.sub.421, nfE4.sub.524, nfE5.sub.1982, nfE5.sub.1515,
nfE5.sub.2144, nfE5.sub.1650, nfE6.sub.1488, nfE6.sub.1792,
nfE6.sub.1650, nfE7.sub.2836, nfE7.sub.1788, nfE7.sub.1710,
nfE7.sub.650, nfE8.sub.2801, nfE8.sub.1785, nfE8.sub.1710,
nfE9.sub.2007, nfE9.sub.1584, nfE9.sub.1540, nfE10.sub.987 and/or
nfE10.sub.1590, or of allelic variants of such nucleic acid
molecules.
[0098] More preferred is an esterase protein encoded by
nfE1.sub.401, nfE2.sub.364, nfE3.sub.421, nfE4.sub.524,
nfE5.sub.1982, nfE5.sub.1515, nfE5.sub.2144, nfE5.sub.1650,
nfE6.sub.1488, nfE6.sub.1792, nfE6.sub.1650, nfE7.sub.2836,
nfE7).sub.788, nfE7.sub.1710, nfE7.sub.650, nfE8.sub.2801,
nfE8.sub.1785, nfE8.sub.1710, nfE9.sub.2007, nfE9.sub.1584,
nfE9.sub.1540, nfE10.sub.197 and/or nfE10.sub.1590, or by an
allelic variant of such nucleic acid molecules. Particularly
preferred arthropod esterase proteins are PfE1.sub.103,
PfE2.sub.121, PfE3.sub.103, PfE4.sub.137, PfE5.sub.505,
PfE5.sub.550, PfE6.sub.550, PfE6.sub.530, PfE7.sub.596,
PfE7.sub.570, PfE8.sub.595, PfE8.sub.570, PfE9.sub.528 and
PfE10.sub.530.
[0099] In one embodiment, a preferred esterase protein of the
present invention is encoded by 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:18, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:24, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:34, SEQ
ID NO:36, SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:60 and/or SEQ ID
NO:67, and, as such, has an amino acid sequence that includes at
least a portion of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID
NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ
ID NO:37, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58
and/or SEQ ID NO:68. Also preferred is a protein encoded by an
allelic variant of a nucleic acid molecule comprising at least a
portion of the above-listed nucleic acid sequences. Particularly
preferred esterase proteins of the present invention include SEQ ID
NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID
NO:19, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ
ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44,
SEQ ID NO:53, SEQ ID
[0100] NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID
NO:73 and/or SEQ ID NO:74. (including, but not limited to, the
proteins consisting of such sequences, fusion proteins and
multivalent proteins) and proteins encoded by allelic variants 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:18, SEQ ID NO:21, SEQ ID NO:23, SEQ ID
NO:24, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:33, SEQ
ID NO:34, SEQ ID NO:36, SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:60
and/or SEQ ID NO:67.
[0101] Another embodiment of the present invention is an isolated
nucleic acid molecule that hybridizes under stringent hybridization
conditions with a C. felis esterase gene. The identifying
characteristics of such a gene are heretofore described. A nucleic
acid molecule of the present invention can include an isolated
natural arthropod esterase gene or a homolog 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
the minimal size that can form a stable hybrid with a C. felis
esterase gene under stringent hybridization conditions.
[0102] 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 subject 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. An isolated
arthropod esterase nucleic acid molecule of the present invention
can be isolated from its natural source or can be produced using
recombinant DNA technology (e.g., polymerase chain reaction (PCR)
amplification, cloning) or chemical synthesis. Isolated esterase
nucleic acid molecules 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 an esterase protein of
the present invention or to form stable hybrids under stringent
conditions with natural gene isolates.
[0103] An arthropod esterase nucleic acid molecule homolog can be
produced using a number of methods known to those skilled in the
art (see, for example, Sambrook et al., ibid.). For example,
nucleic acid molecules can be modified using a variety of
techniques including, but not limited to, classic mutagenesis and
recombinant DNA techniques (e.g., site-directed mutagenesis,
chemical treatment, restriction enzyme cleavage, ligation of
nucleic acid fragments and/or 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 homologs can be selected by hybridization
with a C. felis esterase gene or by screening for 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 an
arthropod esterase protein, hydrolyze .alpha.-napthyl acetate,
hydrolyze the methyl ester group of juvenile hormone and/or blind
to DFP).
[0104] An isolated nucleic acid molecule of the present invention
can include a nucleic acid sequence that encodes at least one
arthropod esterase 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 an arthropod esterase protein.
[0105] A preferred nucleic acid molecule of the present invention,
when administered to an animal, is capable of protecting that
animal from infestation by a hematophagous ectoparasite. As will be
disclosed in more detail below, such a nucleic acid molecule can
be, or can 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 esterase protein (e.g.,
an esterase protein of the present invention), the nucleic acid
molecule being delivered to the animal, for example, by direct
injection (i.e, as a naked nucleic acid) or in a vehicle such as a
recombinant virus vaccine or a recombinant cell vaccine.
[0106] One embodiment of the present invention is an esterase
nucleic acid molecule that hybridizes under stringent hybridization
conditions with nucleic acid molecule nfE1.sub.401 and preferably
with a nucleic acid molecule having nucleic acid sequence SEQ ID
NO:1 and/or SEQ ID NO:3.
[0107] Another embodiment of the present invention is an esterase
nucleic acid molecule that hybridizes under stringent hybridization
conditions with nucleic acid molecule nfE2.sub.364 and preferably
with a nucleic acid molecule having nucleic acid sequence SEQ ID
NO:4 and/or SEQ ID NO:6.
[0108] Another embodiment of the present invention is an esterase
nucleic acid molecule that hybridizes under stringent hybridization
conditions with nucleic acid molecule nfE3.sub.424 and preferably
with a nucleic acid molecule having nucleic acid sequence SEQ ID
NO:7 and/or SEQ ID NO:9. Another embodiment of the present
invention is an esterase nucleic acid molecule that hybridizes
under stringent hybridization conditions with nucleic acid molecule
nfE4.sub.524 and preferably with a nucleic acid molecule having
nucleic acid sequence SEQ ID NO:10 and/or SEQ ID NO:12.
[0109] Another embodiment of the present invention is an esterase
nucleic acid molecule that hybridizes under stringent hybridization
conditions with nucleic acid molecule nfE5.sub.2144 and preferably
with a nucleic acid molecule having nucleic acid sequence SEQ ID
NO:57 and/or SEQ ID NO:59.
[0110] Another embodiment of the present invention is an esterase
nucleic acid molecule that hybridizes under stringent hybridization
conditions with nucleic acid molecule nfE6.sub.1792 and preferably
with a nucleic acid molecule having nucleic acid sequence SEQ ID
NO:18 and/or SEQ ID NO:20.
[0111] Another embodiment of the present invention is an esterase
nucleic acid molecule that hybridizes under stringent hybridization
conditions with nucleic acid molecule nfE7.sub.2836 and preferably
with a nucleic acid molecule having nucleic acid sequence SEQ ID
NO:24 and/or SEQ ID NO:26.
[0112] Another embodiment of the present invention is an esterase
nucleic acid molecule that hybridizes under stringent hybridization
conditions with nucleic acid molecule nfE8.sub.280, and preferably
with a nucleic acid molecule having nucleic acid sequence SEQ ID
NO:30 and/or SEQ ID NO:32. Another embodiment of the present
invention is an esterase nucleic acid molecule that hybridizes
under stringent hybridization conditions with nucleic acid molecule
nfE9.sub.2007 and preferably with a nucleic acid molecule having
nucleic acid sequence SEQ ID NO:36 and/or SEQ ID NO:38.
[0113] Another embodiment of the present invention is an esterase
nucleic acid molecule that hybridizes under stringent hybridization
conditions with nucleic acid molecule nfE10.sub.1987 and preferably
with a nucleic acid molecule having nucleic acid sequence SEQ ID
NO:67 and/or SEQ ID NO:69.
[0114] Comparison of nucleic acid sequence SEQ ID NO:1 (i.e., the
nucleic acid sequence of nfE1.sub.401) with nucleic acid sequences
reported in GenBank indicates that SEQ ID NO:1 showed no
identifiable identity with any sequence reported in GenBank.
[0115] Comparison of nucleic acid sequence SEQ ID NO:4 (i.e., the
coding strand of nucleic acid sequence of nfE2.sub.364) with
nucleic acid sequences reported in GenBank indicates that SEQ ID
NO:4 showed the most homolog, i.e., about 43% identity, between SEQ
ID NO:4 and a H. virescens juvenile hormone esterase gene.
[0116] Comparison of nucleic acid sequence SEQ ID NO:7 (i.e., the
coding strand of nucleic acid sequence of nfE3.sub.421) with
nucleic acid sequences reported in GenBank indicates that SEQ ID
NO:7 showed the most homolog, i.e., about 53% identity, between SEQ
ID NO:7 and a Torpedo marmorata acetylcholinesterase gene.
[0117] Comparison of nucleic acid sequence SEQ ID NO:10 (i.e., the
coding strand of nucleic acid sequence of nfE4.sub.524) with
nucleic acid sequences reported in GenBank indicates that SEQ ID
NO:10 showed the most homolog, i.e., about 47% identity, between
SEQ ID NO:10 and an Anas platyrhynos thioesterase B gene.
[0118] Comparison of nucleic acid sequence SEQ ID NO:57 (i.e., the
coding strand of nucleic acid sequence of nfE5.sub.2144) with
nucleic acid sequences reported in GenBank indicates that SEQ ID
NO:57 showed the most homolog, i.e., about 41% identity, between
SEQ ID NO:57 and a esterase mRNA from Myzus persicae.
[0119] Comparison of nucleic acid sequence SEQ ID NO:18 (i.e., the
coding strand of nucleic acid sequence of nfE6.sub.1792) with
nucleic acid sequences reported in GenBank indicates that SEQ ID
NO:18 showed the most homolog, i.e., about 41% identity, between
SEQ ID NO:18 and a esterase gene from Myzus persicae.
[0120] Comparison of nucleic acid sequence SEQ ID NO:24 (i.e., the
coding strand of nucleic acid sequence of nfE7.sub.2836) with
nucleic acid sequences reported in GenBank indicates that SEQ ID
NO:24 showed the most homolog, i.e., about 48% identity, between
SEQ ID NO:24 and an Anas platychyncos thioesterase B gene.
[0121] Comparison of nucleic acid sequence SEQ ID NO:30 (i.e., the
coding strand of nucleic acid sequence of nfE8.sub.2801) with
nucleic acid sequences reported in GenBank indicates that SEQ ID
NO:30 showed the most homolog, i.e., about 46% identity, between
SEQ ID NO:30 and a Mus musculus carboxyl ester lipase gene.
[0122] Comparison of nucleic acid sequence SEQ ID NO:36 (i.e., the
coding strand of nucleic acid sequence of nfE9.sub.2007) with
nucleic acid sequences reported in GenBank indicates that SEQ ID
NO:36 showed the most homolog, i.e., about 47% identity, between
SEQ ID NO:36 and a hamster mRNA for CE precursor gene.
[0123] Comparison of nucleic acid sequence SEQ ID NO:67 with
nucleic acid sequences reported in GenBank indicates that SEQ ID
NO:67 showed the most homology, i.e., about 48% identity, between
SEQ ID NO:67 and a Lucilia cuprina alpha esterase gene (genembl
#U56636) gene.
[0124] Preferred arthropod esterase nucleic acid molecules include
nucleic acid molecules having a nucleic acid sequence that is at
least about 55%, preferably at least about 60%, more preferably at
least about 65%, more preferably at least about 70%, more
preferably at least about 75%, more preferably at least about 80%,
more preferably at least about 90%, and even more preferably at
least about 95% identical to 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:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22,
SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID
NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:33, SEQ
ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:51,
SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:60, SEQ ID
NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ
ID NO:72, SEQ ID NO:76 and/or a nucleic acid molecule encoding a
protein comprising amino acid sequence SEQ ID NO:74.
[0125] 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:17, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:21,
SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID
NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:32, SEQ
ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:38,
SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59, SEQ ID
NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:70, SEQ
ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic acid molecule
encoding a protein comprising amino acid sequence SEQ ID NO:74,
that is capable of hybridizing to a C. felis esterase gene of the
present invention, as well as allelic variants thereof. A more
preferred nucleic acid molecule includes the 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:17, SEQ ID NO:18, SEQ ID NO:20, SEQ
ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26,
SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID
NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ
ID NO:38, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:57, SEQ ID NO:59,
SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:67, SEQ ID NO:69, SEQ ID
NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:76 and/or a nucleic
acid molecule encoding a protein comprising amino acid sequence SEQ
ID NO:74, as well as allelic variants thereof. 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. Particularly preferred nucleic acid molecules
include nfE1.sub.401, nfE2.sub.364, nfE3.sub.421, nfE4.sub.524,
nfE5.sub.1982, nfE5.sub.1515, nfE5.sub.2144, nfE5.sub.1650,
nfE6.sub.1488, nfE6.sub.1792, nfE6.sub.1650, nfE7.sub.2836,
nfE7.sub.1788, nfE7.sub.1710, nfE7.sub.650, nfE8.sub.2801,
nfE8.sub.1785, nfE8.sub.1710, nfE9.sub.2007, nfE9.sub.1584,
nfE9.sub.1540, nfE10.sub.1987 and nfE10.sub.1590.
[0126] The present invention also includes a nucleic acid molecule
encoding a protein having at least a portion of SEQ ID NO:2, SEQ ID
NO:5, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:19, SEQ ID
NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:40, SEQ
ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:53,
SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:68, SEQ ID
NO:73 and/or SEQ ID NO:74, 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.
[0127] Knowing the nucleic acid sequences of certain arthropod
esterase 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 esterase nucleic acid molecules from other
arthropods. 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 or DNA; 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 molecule include flea pre-pupal,
3rd instar or adult cDNA libraries as well as genomic DNA
libraries. Similarly, preferred DNA sources to screen or from which
to amplify nucleic acid molecules include flea pre-pupal, 3rd
instar or adult cDNA and genomic DNA. Techniques to clone and
amplify genes are disclosed, for example, in Sambrook et al.,
ibid.
[0128] 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 arthropod esterase genes or other
arthropod esterase 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. Minimal size characteristics are disclosed herein. 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
esterase 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.
[0129] 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 manipulation of arthropod esterase
nucleic acid molecules of the present invention.
[0130] 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, endoparasite, 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.
[0131] 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 which
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 which function in
bacterial, yeast, 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 promoters (such as Sindbis virus subgenomic
promoters), antibiotic resistance gene, baculovirus, Heliothis zea
insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other
poxvirus, adenovirus, cytomegalovirus (such as intermediate early
promoters), 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
arthropods, such as, C. felis.
[0132] 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
nfE1.sub.401, nfE2.sub.364, nfE3.sub.421, nfE4.sub.524,
nfE5.sub.1982, nfE5.sub.515, nfE5.sub.2144, nfE5.sub.1650,
nfE6.sub.1488, nfE6.sub.1792, nfE6.sub.1650, nfE7.sub.2836,
nfE7.sub.1788, nfE7.sub.1710, nfE7.sub.650, nfE8.sub.2801,
nfE8.sub.1785, nfE8.sub.1710, nfE9.sub.2007, nfE9.sub.1584,
nfE9.sub.1540, nfE10.sub.1987 and/or nfE10.sub.1590 Particularly
preferred recombinant molecules of the present invention include
pCro-nfE6.sub.1488, pTrc-nfE7.sub.650, pTrc-nfE7.sub.1710,
pTrc-nfE8.sub.1710, pTrc-nfE5.sub.1650, pTrc-nfE9.sub.1540,
pFB-nfE6.sub.1679, pVL-nfE7.sub.1802, pVL-nfE8.sub.1792 and
pVL-nfE9.sub.1600, the production of which are described in the
Examples section.
[0133] Recombinant molecules of the present invention may also (a)
contain secretory signals (i.e., signal segment nucleic acid
sequences) to enable an expressed arthropod 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, as well as natural signal sequences. 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. 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.
[0134] 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. 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 arthropod esterase nucleic acid
molecules disclosed herein. Particularly preferred nucleic acid
molecules with which to transform a cell include, nfE1.sub.401,
nfE2.sub.364, nfE3.sub.421, nfE4.sub.524, nfE5.sub.1982,
nfE5.sub.1515, nfE5.sub.2144, nfE5.sub.1650, nfE6.sub.1488,
nfE6.sub.1792, nfE6.sub.1650, nfE7.sub.2836, nfE7.sub.1788,
nfE7.sub.1710, nfE7.sub.650, nfE8.sub.2801, nfE8.sub.1785,
nfE8.sub.1710, nfE9.sub.2007, nfE9).sub.584, nfE9.sub.1540,
nfE10.sub.1987 and/or nfE10.sub.1590.
[0135] 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 arthropod esterase 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, 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,
Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia,
BHK (baby hamster kidney) cells, MDCK cells (normal dog kidney cell
line for canine herpesvirus cultivation), CRFK cells (normal cat
kidney cell line for feline herpesvirus cultivation), 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.X3987 and SR-11
.sub.X4072; 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.
[0136] 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. 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.
[0137] A recombinant molecule of the present invention is a
molecule that can include at least one of any nucleic acid molecule
heretofore described operatively linked to at least one of any
transcription control sequence capable of effectively regulating
expression of the nucleic acid molecule(s) in the cell to be
transformed, examples of which are disclosed herein. Particularly
preferred recombinant molecules include pCro-nfE6.sub.1488,
pTrc-nfE7.sub.650, pTrc-nfE7.sub.1710, pTrc-nfE8.sub.1710,
pTrc-nfE5.sub.1650, pTrc-nfE9.sub.1540, pFB-nfE6.sub.1679,
pVL-nfE7.sub.1802, pVL-nfE8.sub.1792 and pVL-nfE9.sub.1600.
[0138] 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 transform cells are disclosed herein. Particularly
preferred recombinant cells include E. coli:pCro-nfE6.sub.1488, E.
coli:pTrc-nfE7.sub.1710, E coli:pTrc-nfE7.sub.650, E.
coli:pTrc-nfE8.sub.1710, E. coli:pTrc-nfE5.sub.1650, E.
coli:pTrc-nfF9.sub.1540, S. frugiperda:pVL-nfE7 .sub.102, S.
frugiperda:pVL-nfE8.sub.1792, S. frugiperda:pVL-nfE9.sub.1600 and
S. frugiperda:pFB-nfE6.sub.1679. Details regarding the production
of these recombinant cells are disclosed herein.
[0139] Recombinant cells of the present invention can also be
co-transformed with one or more recombinant molecules including
arthropod esterase 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).
[0140] 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.
[0141] Isolated esterase 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 an arthropod esterase
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.
[0142] 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. 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.
[0143] The present invention also includes isolated (i.e., removed
from their natural milieu) antibodies that selectively bind to an
arthropod esterase protein of the present invention or a mimetope
thereof (i.e., anti-arthropod esterase antibodies). As used herein,
the term "selectively binds to" an esterase 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.
An anti-arthropod esterase antibody preferably selectively binds to
an arthropod esterase protein in such a way as to reduce the
activity of that protein.
[0144] Isolated antibodies of the present invention can include
antibodies in a bodily fluid such as, but not limited to, serum),
or antibodies that have been purified to varying degrees.
Antibodies of the present invention can be polyclonal or
monoclonal, functional equivalents such as antibody fragments and
genetically-engineered antibodies, including single chain
antibodies or chimeric antibodies that can bind to more than one
epitope.
[0145] 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 arthropod esterase 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.
[0146] 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 arthropods 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
hematophagous ectoparasites such as those discloses herein, in
order to directly kill such hematophagous ectoparasites. 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.
[0147] One embodiment of the present invention is a therapeutic
composition that, when administered to an animal in an effective
manner, is capable of protecting that animal from infestation by
hematophagous ectoparasite. Therapeutic compositions of the present
invention include at least one of the following protective
compounds: an isolated hematophagous arthropod esterase protein
(including a peptide); a mimetope of such a protein; an isolated
nucleic acid molecule that hybridizes under stringent hybridization
conditions with a Ctenocephalides felis esterase gene; an isolated
antibody that selectively binds to an hematophagous arthropod
esterase protein; and inhibitors of hematophagous arthropod
esterase activity (including esterase substrate analogs). As used
herein, a protective compound refers to a compound that, when
administered to an animal in an effective manner, is able to treat,
ameliorate, and/or prevent disease caused by an arthropod of the
present invention. Preferred arthropods to target are heretofore
disclosed. Examples of proteins, nucleic acid molecules, antibodies
and inhibitors of the present invention are disclosed herein.
[0148] A preferred therapeutic composition of the present invention
includes at least one of the following protective compounds: an
isolated hematophagous ectoparasite carboxylesterase protein
(including a peptide); a mimetope of such a protein; an isolated
hematophagous ectoparasite carboxylesterase nucleic acid molecule
that hybridizes under stringent hybridization conditions with a
Ctenocephalides felis carboxylesterase gene; an isolated antibody
that selectively binds to a hematophagous ectoparasite
carboxylesterase protein; and an inhibitor of carboxylesterase
activity identified by its ability to inhibit the activity of a
flea carboxylesterase (including a substrate analog).
[0149] Suitable inhibitors of esterase activity are compounds that
interact directly with an esterase protein's active site, thereby
inhibiting that esterase's activity, usually by binding to or
otherwise interacting with or otherwise modifying the esterase's
active site. Esterase inhibitors can also interact with other
regions of the esterase protein to inhibit esterase activity, for
example, by allosteric interaction. Inhibitors of esterases are
usually relatively small compounds and as such differ from
anti-esterase antibodies. Preferably, an esterase inhibitor of the
present invention is identified by its ability to bind to, or
otherwise interact with, a flea esterase protein, thereby
inhibiting the activity of the flea esterase.
[0150] Esterase 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.
Esterase inhibitors can also be used to identify preferred types of
arthropod esterases to target using compositions of the present
invention, for example by affinity chromatography. Preferred
esterase inhibitors of the present invention include, but are not
limited to, flea esterase substrate analogs, and other molecules
that bind to a flea esterase (e.g., to an allosteric site) in such
a manner that esterase activity of the flea esterase is inhibited;
examples include, but are not limited to, juvenile hormone analogs
and cholinesterase inhibitors as well as other neural transmission
inhibitors. An esterase substrate analog refers to a compound that
interacts with (e.g., binds to, associates with, modifies) the
active site of an esterase protein. A preferred esterase substrate
analog inhibits esterase activity. Esterase substrate analogs can
be of any inorganic or organic composition, and, as such, can be,
but are not limited to, peptides, nucleic acids, and peptidomimetic
compounds. Esterase substrate analogs can be, but need not be,
structurally similar to an esterase's natural substrate as long as
they can interact with the active site of that esterase protein.
Esterase substrate analogs can be designed using computer-generated
structures of esterase proteins of the present invention or
computer structures of esterases' natural substrates. 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 by affinity chromatography techniques using the
corresponding binding partner, (e.g., a flea esterase). A preferred
esterase substrate analog is a peptidomimetic compound (i.e., a
compound that is structurally and/or functionally similar to a
natural substrate of an esterase of the present invention,
particularly to the region of the substrate that interacts with the
esterase active site, but that inhibits esterase activity upon
interacting with the esterase active site).
[0151] Esterase peptides, mimetopes and substrate analogs, as well
as other protective compounds, can be used directly as compounds in
compositions of the present invention to treat animals as long as
such compounds are not harmful to the animals being treated.
[0152] The present invention also includes a therapeutic
composition comprising at least one arthropod esterase-based
compound of the present invention in combination wraith at least
one additional compound protective against hematophagous
ectoparasite infestation. Examples of such compounds are disclosed
herein.
[0153] In one embodiment, a therapeutic composition of the present
invention can be used to protect an animal from hematophagous
ectoparasite infestation by administering such composition to a
hematophagous ectoparasite, such as to a flea, in order to prevent
infestation. Such administration could be oral, 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 hematophagous ectoparasite,
such as a flea, can ingest therapeutic compositions, or products
thereof, present in the blood of a host animal that has been
administered a therapeutic composition of the present
invention.
[0154] Compositions of the present invention can be administered to
any animal susceptible to hematophagous ectoparasite infestation
(i.e., a host animal), including warm-blooded animals. Preferred
animals to treat include mammals and birds, with cats, dogs,
humans, cattle, chinchillas, ferrets, goats, mice, minks, rabbits,
raccoons, rats, sheep, squirrels, swine, chickens, ostriches, quail
and turkeys as well as other furry animals, pets, zoo animals, work
animals and/or food animals, being more preferred. Particularly
preferred animals to protect are cats and dogs.
[0155] In accordance with the present invention, a host animal
(i.e., an animal that is or is capable of being infested with a
hematophagous ectoparasite) is treated by administering to the
animal a therapeutic composition of the present invention in such a
manner that the composition itself (e.g., an esterase inhibitor, an
esterase synthesis suppressor (i.e., a compound that decreases the
production of esterase in the hematophagous ectoparasite), an
esterase mimetope, or an anti-esterase antibody) or a product
generated by the animal in response to administration of the
composition (e.g., antibodies produced in response to
administration of an arthropod esterase protein or nucleic acid
molecule, or conversion of an inactive inhibitor "prodrug" to an
active esterase inhibitor) ultimately enters the hematophagous
ectoparasite. A host animal is preferably treated in such a way
that the compound or product thereof enters the blood stream of the
animal. Hematophagous ectoparasites are then exposed to the
composition or product when they feed from the animal. For example,
flea esterase 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. In another
embodiment, when a host animal is administered an arthropod
esterase protein or nucleic acid molecule, the treated animal
mounts an immune response resulting in the production of antibodies
against the esterase (i.e., anti-esterase antibodies) which
circulate in the animal's blood stream and are taken up by
hematophagous ectoparasites upon feeding. Blood taken up by
hematophagous ectoparasites enters the hematophagous ectoparasites
where compounds of the present invention, or products thereof, such
as anti-esterase antibodies, esterase inhibitors, esterase
mimetopes and/or esterase synthesis suppressors, interact with, and
reduce esterase activity in the hematophagous ectoparasite.
[0156] The present invention also includes the ability to reduce
larval hematophagous ectoparasite infestation in that when
hematophagous ectoparasites 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 hematophagous ectoparasite
are excreted by the hematophagous ectoparasite in feces, which is
subsequently ingested by hematophagous ectoparasite larvae. In
particular, it is of note that flea larvae obtain most, if not all,
of their nutrition from flea feces.
[0157] In accordance with the present invention, reducing esterase
activity in a hematophagous ectoparasite can lead to a number of
outcomes that reduce hematophagous ectoparasite burden on treated
animals and their surrounding environments. Such outcomes include,
but are not limited to, (a) reducing the viability of hematophagous
ectoparasites that feed from the treated animal, (b) reducing the
fecundity of female hematophagous ectoparasites that feed from the
treated animal, (c) reducing the reproductive capacity of male
hematophagous ectoparasites that feed from the treated animal, (d)
reducing the viability of eggs laid by female hematophagous
ectoparasites that feed from the treated animal, (e) altering the
blood feeding behavior of hematophagous ectoparasites that feed
from the treated animal (e.g., hematophagous ectoparasites take up
less volume per feeding or feed less frequently), (f) reducing the
viability of hematophagous ectoparasite larvae, for example due to
the feeding of larvae from feces of hematophagous ectoparasites
that feed from the treated animal and so- (g) altering the
development of hematophagous ectoparasite larvae (e.g., by
decreasing feeding behavior, inhibiting growth, inhibiting (e.g.,
slowing or blocking) molting, and/or otherwise inhibiting
maturation to adults).
[0158] Therapeutic compositions of the present invention also
include excipients in which protective compounds are formulated. An
excipient can be any material 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, dog serum albumin, cat serum albumin, preservatives, etc.,
to which sterile water or saline can be added prior to
administration.
[0159] 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,
cytokinies, chemokines, and compounds that induce the production of
cytokines and chemokines (e.g., granulocyte macrophage colony
stimulating factor (GM-CSF), 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.
[0160] 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.
[0161] 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).
[0162] A preferred controlled release formulation of the present
invention is capable of releasing a composition of the present
invention into the blood of an animal at a constant rate sufficient
to attain therapeutic dose levels of the composition to protect an
animal from hematophagous ectoparasite infestation. The therapeutic
composition is preferably released over a period of time ranging
from about 1 to about 12 months. A preferred 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.
[0163] Acceptable protocols to administer therapeutic compositions
of the present invention 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 is from about 1 microgram (.mu.g) 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 and intramuscular routes.
[0164] 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 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).
[0165] A 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 naked nucleic acid vaccine of the present invention
can comprise one or more nucleic acid molecules of the present
invention in the form of, for example, a bicistronic recombinant
molecule having, for example one or more internal ribosome entry
sites. Preferred naked nucleic acid 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, and retroviruses, with those based on
alphaviruses (such as Sindbis or Semliki virus), species-specific
herpesviruses and species-specific poxviruses being particularly
preferred. Any suitable transcription control sequence can be used,
including those disclosed as suitable for protein production.
Particularly preferred transcription control sequence include
cytomegalovirus intermediate 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 "strong" poly(A)
sequences are also preferred.
[0166] Naked nucleic acid vaccines of the present invention can be
administered in a variety of ways, with intramuscular,
subcutaneous, intradermal, transdermal, intranasal and oral routes
of administration being preferred. A preferred single dose of a
naked nucleic acid vaccines ranges from about 1 nanogram (ng) to
about 100 .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. Naked DNA of the present
invention can be contained in an aqueous excipient (e.g., phosphate
buffered saline) alone or a carrier (e.g., lipid-based
vehicles).
[0167] 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-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, 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 is disclosed in PCT
Publication No. WO 94/17813, by Xiong et al., published Aug. 18,
1994, which is incorporated by reference herein in its
entirety.
[0168] 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
hematophagous ectoparasite infestation. For example, a recombinant
virus vaccine comprising an arthropod CE 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 hematophagous ectoparasite 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.7
virus plaque forming units (pfu) per kilogram body weight of the
animal. Administration protocols are similar to those described
herein for protein-based vaccines, with subcutaneous,
intramuscular, intranasal and oral administration routes being
preferred.
[0169] 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.
[0170] Preferred recombinant cells for this embodiment include
Salmonella, E. coli, Listeria, Mycobacterium, S. frugiperda, yeast,
(including Saccharomyces cerevisiae), 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.
[0171] The efficacy of a therapeutic composition of the present
invention to protect an animal from hematophagous ectoparasite
infestation can be tested in a variety of ways including, but not
limited to, detection of anti-arthropod esterase 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 hematophagous ectoparasites to
determine whether, for example, the feeding, fecundity or viability
of hematophagous ectoparasites feeding from the treated animal is
disrupted. Challenge studies can include attachment of chambers
containing hematophagous ectoparasites onto the skin of 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.
[0172] One preferred embodiment of the present invention is the use
of arthropod protective compounds, such as proteins, mimetopes,
nucleic acid molecules, antibodies and inhibitory compounds of the
present invention, to protect an animal from hematophagous
ectoparasite, and particularly flea, infestation. Preferred
protective compounds of the present invention include, but are not
limited to, C. felis esterase nucleic acid molecules, C. felis
esterase proteins and mimetopes thereof, anti-C. felis esterase
antibodies, and inhibitors of C. felis esterase activity. More
preferred protective compounds of the present invention include,
but are not limited to, CE or JHE formulations of the present
invention, C. felis CE nucleic acid molecules, C. felis CE proteins
and mimetopes thereof, anti-flea CE antibodies, anti-flea JHE
antibodies, inhibitors of C. felis CE activity and inhibitors of
flea JHE activity. Additional protection may be obtained by
administering additional protective compounds, including other
proteins, mimetopes, nucleic acid molecules, antibodies and
inhibitory compounds, as disclosed herein.
[0173] One therapeutic composition of the present invention
includes an inhibitor of arthropod esterase activity, i.e., a
compound capable of substantially interfering with the function of
an arthropod esterase susceptible to inhibition by an inhibitor of
arthropod esterase activity. An inhibitor of esterase activity can
be identified using arthropod esterase proteins of the present
invention. One embodiment of the present invention is a method to
identify a compound capable of inhibiting esterase activity of an
arthropod. Such a method includes the steps of (a) contacting
(e.g., combining, mixing) an isolated flea esterase protein,
preferably a C. felis esterase protein of the present invention,
with a putative inhibitory compound under conditions in which, in
the absence of the compound, the protein has esterase activity, and
(b) determining if the putative inhibitory compound inhibits the
esterase activity. Putative inhibitory compounds to screen include
small organic molecules, antibodies (including mimetopes thereof)
and substrate analogs. Methods to determine esterase activity are
known to those skilled in the art; see, for example, the Examples
section of the present application.
[0174] The present invention also includes a test kit to identify a
compound capable of inhibiting esterase activity of an arthropod.
Such a test kit includes an isolated flea esterase protein,
preferably a C. felis esterase protein, having esterase activity
and a means for determining the extent of inhibition of esterase
activity in the presence of (i.e., effected by) a putative
inhibitory compound. Such compounds are also screened to identify
those that are substantially not toxic in host animals.
[0175] Esterase inhibitors isolated by such a method, and/or test
kit, can be used to inhibit any esterase that is susceptible to
such an inhibitor. Preferred esterase proteins to inhibit are those
produced by arthropods. A particularly preferred esterase inhibitor
of the present invention is capable of protecting an animal from
hematophagous ectoparasite infestation. Effective amounts and
dosing regimens can be determined using techniques known to those
skilled in the art.
[0176] The following examples are provided for the purposes of
illustration and are not intended to limit the scope of the present
invention.
EXAMPLES
[0177] It is to be noted that the Examples include a number of
molecular biology, microbiology, immunology and biochemistry
techniques considered to be known to those skilled in the art.
Disclosure of such techniques can be found, for example, in
Sambrook et al., ibid., Borovsky, Arch Insect Biochem. and Phys.,
7:187-210, 1988, and related references.
Example 1
[0178] This example describes labeling of proteases and esterases
with radiolabeled diisopropylfuorophosphate.
[0179] Tissue samples were isolated from unfed or bovine blood-fed
1 st instar Ctenocephalides felis flea larvae; bovine blood-fed or
cat blood-fed 3rd instar Ctenocephalides felis flea larvae; bovine
blood-fed or cat blood-fed Ctenocephalides felis prepupal flea
larvae; bovine blood-fed or cat blood-fed adult Ctenocephalides
felis flea midgut tissue, and whole unfed, bovine blood-fed or cat
blood-fed adult Ctenocephalides felis fleas. The 1 st instar, 3rd
instar, prepupal and adult midgut tissues were then homogenized by
freeze-fracture and sonicated in a Tris buffer comprising 50 mM
Tris, pH 8.0 and 100 mM CaCl.sub.2. The whole adult flea sample was
then homogenized by freeze-fracture and ground with a microtube
mortar and pestle. The extracts were centrifuged at about
14,000.times.g for 20 minutes (min.) and the soluble material
recovered. The soluble material was then diluted to a final
concentration of about 1 to about 1.2 tissue equivalents per
microliter (.mu.l) of Tris buffer. Each sample was labeled with
[1,3-.sup.3H]-diisopropylfluorophosphate (3H-DFP) (available from
DuPont-NEN, Wilmington, Del.) using the method generally described
in Borovsky, ibid. About 20 tissue equivalents of each tissue
sample were mixed with about 1 .mu.Ci of .sup.3H-DFP and incubated
for about 18 hours at 4.degree. C. Proteins contained in each
sample were then resolved using a 14% Tris-glycine sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (available
from Novex, San Diego, Calif.) under reducing conditions. The gel
was soaked in Entensify (available from DuPont-NEN) according to
manufacturers instructions, and exposed to X-ray film (available
from Kodak X-0mat AR, Rochester, N.Y.) for about 3 days at
-70.degree. C.
[0180] Analysis of the resulting autoradiogram (shown in FIG. 1)
indicated that tissue samples from 3rd instar, prepupal larvae and
whole adult flea contained proteins that labeled with DFP, having a
molecular weight (MW) of about 60 kilodalton (kD). No proteins of
this MW were labeled in tissue samples from unfed or fed 1st instar
larvae and adult midgut. The results indicated preferred tissue
distribution and stage-specific expression of DFP-labeled serine
esterases in fleas.
Example 2
[0181] This example describes the identification of general CE
activity in flea tissue extracts.
[0182] Tissue samples and soluble extracts were prepared as
described above in Example 1, except not labelled, from unfed (UF)
and bovine blood-fed 1st instar flea larvae, bovine blood-fed 3rd
instar flea larvae, bovine blood-fed prepupal flea larvae, unfed
whole adult fleas, cat blood-fed adult (ACF) whole fleas, cat
blood-fed adult fleas that have had their heads and midguts removed
(referred to herein as fed adult partial fleas), unfed adult flea
midguts and cat blood-fed adult flea midguts. About 5 tissue
equivalents of each tissue were assayed for general CE activity
using the following method. Tissue samples of about 5 .mu.l were
added to separate wells of flat-bottomed microtiter plate
(available from Becton Dickinson, Lincoln Park, N.J.). A control
well was prepared by adding about 5 .mu.l of Tris buffer to an
empty well of the plate. About 95 .mu.l of 25 mM Tris-HCl (pH 8.0)
was then added to each sample to increase the volume in each well
to about 100 ill. About 100 .mu.l of 0.25 mM .alpha.-napthyl
acetate (available from Sigma, St. Louis, Mo.) dissolved in 25 mM
Tris-HCl (pH 8.0) was then added to each well. The plate was then
incubated for about 15 min. at 37.degree. C. Following the
incubation, about 40 .mu.l of 0.3% Fast Blue salt BN (tetrazotized
o-dianisidine; available from Sigma) dissolved in 3.3% SDS in water
was added to each well.
[0183] The microtiter plate was then analyzed using a Cambridge
Technology, Inc. (Watertown, Pa.) model 7500 Microplate Reader set
to 590 nm. The absorbance value for the control sample was
subtracted from absorbance values of experimental samples, such
that the background value was zero.
[0184] The results shown in FIG. 2 indicated that general CE
activity was detected in all tissue samples. The level of activity
varied, with unfed and fed 1st instar larvae, unfed adult flea
midguts, and fed adult flea midguts having relatively lower
activity than in the other tissues. Thus, the results indicated
preferred tissue distribution and stage-specific expression of
general CE activity in fleas.
Example 3
[0185] This example describes the determination of general CE
activity using isoelectric focusing (IEF)-PAGE and non-reducing
SDS-PAGE.
[0186] A. Non-Reducing SDS-PAGE.
[0187] Soluble extracts from unfed and bovine blood-fed 1st instar
flea larvae, bovine blood-fed 3rd instar flea larvae, bovine
blood-fed prepupal flea larvae, bovine blood-fed adult (ABF) whole
fleas and cat blood-fed adult whole fleas were prepared using the
method described in Example 1. Each soluble extract sample was
combined with SDS sample buffer (available from Novex) and proteins
in the samples were resolved by gel electrophoresis using 14%
Tris-glycine SDS electrophoresis gels (available from Novex). The
gels were run at room temperature for about 1 hour at 200 volts.
After electrophoresis, the gels were soaked for about for 30
minutes in 50 mM Tris, pH 8.0, containing 2.5% Triton X-100 to
renature the proteins. The gels were then soaked in 50 mM Tris, pH
8.0, for about 5 minutes and then stained for about 5 min. in 50
milliliters (ml) of 25 mM Tris, pH 8.0, containing 50 mg Fast blue
salt BN and 10 mg .alpha.-napthyl acetate (dissolved in 1 ml
acetone). Once protein was detected on the stained gels, the gels
were rinsed with water and photographed.
[0188] B. IEF-PAGE.
[0189] Soluble extracts from unfed and bovine blood-fed 1st instar
flea larvae, bovine blood-fed 3rd instar flea larvae, bovine
blood-fed prepupal flea larvae, unfed and cat blood-fed whole
fleas, cat blood-fed adult partial fleas and cat blood-fed adult
midguts were prepared as described above in Section A. The extracts
were each combined with IEF sample buffer pH 3-7 (available from
Novex) and loaded onto pH 3-7 IEF electrophoresis gels (available
from Novex). The gels were electrophoresed at room temperature
first for about 1 hour at about 100 volts, then for about 1 hour at
about 200 volts, and then for about 30 min. at about 500 volts.
Following electrophoresis, the gels were soaked in 25 mM Tris
buffer, pH 8.0, for about 5 min. and then stained for about 15 min.
in 50 ml of 25 mM Tris buffer, pH 8.0, containing 50 mg Fast blue
salt BN and 10 mg .alpha.-napthyl acetate (dissolved in 1 ml
acetone). Once protein was detected on the stained gels, the gels
were rinsed with water and photographed.
[0190] C. Results.
[0191] The results from gel electrophoresis experiments described
above in Sections A and B are shown in FIGS. 3 and 4. The results
indicated that certain flea tissues contain proteins having MW's of
from about 60 to about 70 kD and native pI values of from about 4.7
to about 5.2 that have CE activity. In particular, CE activity was
identified in prepupal larvae and fed adult flea extracts resolved
by non-reduced SDS-PAGE. No CE activity was identified in unfed and
fed 1st instar larvae or fed 3rd instar larvae extracts (see FIG.
3). When extracts were resolved by native IEF-PAGE, CE activity was
identified in fed 3rd instar larvae, prepupal larvae, unfed and fed
whole adult flea, and fed adult partial flea extracts (see FIG. 4,
lanes 3-7)). No CE activity was identified in unfed or fed 1st
instar larvae, or in fed adult flea midgut extracts (see FIG. 4,
lanes 1, 2, and 8).
Example 4
[0192] This example describes the purification of CE protein from
prepupal flea larvae.
[0193] About 15,000 bovine blood-fed prepupal flea larvae were
collected and the larvae were homogenized in TBS by sonication in
50 ml Oak Ridge centrifuge tubes (available from Nalgene Co.,
Rochester, N.Y.) by sonicating 4 times 20 seconds each at a setting
of 5 of a model W-380 Sonicator (available from Heat
Systems-Ultrasonics, Inc.). The sonicates were clarified by
centrifugation at 18,000 RPM for 30 minutes to produce an extract.
Soluble protein in the extract was removed by aspiration and
diluted to a volume of about 20 ml in TBS (equivalent to about 1
larva per .mu.l TBS). The extract was then added to a column
containing about 5 ml of p-aminobenzamidine linked to agarose beads
(available from Sigma, St. Louis, Mo.) and incubated overnight at
4.degree. C. The column was then washed with about 30 ml TBS to
remove unbound protein. The collected unbound protein was then
concentrated to a volume of about 20 ml using a Macrosep 10
centrifugal protein concentrator (Filtron Technology Corp.,
Northborough, Mass.) and filtered sequentially through a 1 .mu.m
syringe filter and then through a 0.2 .mu.m syringe filter to
clarify the sample for chromatography.
[0194] Aliquots of about 0.5 ml were loaded onto a 20 ml Superdex
200 HR gel filtration column (available from Pharmacia, Piscataway,
N.J.) equilibrated in TBS, operated on a BioLogic liquid
chromatography system (available from BioRad, Burlingame, Calif.).
About 1 ml fractions were then collected. Repetitive runs were
performed until about 30 ml of each fraction was collected. The
fractions were analyzed for CE activity using the assay described
above in Example 2. In preparation for cation exchange
chromatography, fractions having CE activity (V.sub.e=16-18 ml)
were combined and dialyzed against about 2 liters of 20 mM MES
buffer (2-(N-morpholino)ethanesulfonic acid), pH 6.0, containing 10
mM NaCl, for about 1.5 hours, and then against about 1 liter of the
same buffer overnight at 4.degree. C. Prior to loading onto the
cation exchange chromatography column, the sample was again
filtered through a 0.2 .mu.m syringe filter to remove precipitated
proteins. The sample was then applied to a Bio-Scale S2 cation
exchange column (available from BioRad) at a rate of about 0.5
ml/min. The column was washed with MES buffer until all unbound
protein was removed. Protein bound to the column was then eluted
with a linear gradient from 10 mM to 1 M NaCl in 20 mM MES buffer,
pH 6. Fractions were assayed for CE activity using the assay
described above in Example 2. The results indicated that CE
activity was not retained on the cation exchange column using the
above conditions, and all of the activity was found in the
flow-through fractions.
[0195] Fractions containing CE activity were pooled and adjusted to
pH 7 using 0.5 M Tris, pH 8.0, in preparation for anion exchange
chromatography. The pooled fractions were then loaded onto a 4.5
mm.times.50 mm Poros 10 HQ anion exchange chromatography column
(available from PerSeptive Biosystems, Cambridge, Mass.)
equilibrated in 25 mM Tris buffer, pH 6.8. The column was washed
with the loading buffer, and bound proteins were eluted with a
linear gradient of 0 to 1 M NaCl in 25 mM Tris buffer, pH 6.8.
Fractions were tested for CE activity using the assay described
above in Example 2. The results indicated that CE activity was
eluted at about 170 mM NaCl. Fractions containing CE activity were
pooled and diafiltered into TBS.
Example 5
[0196] This example describes the determination of N-terminal amino
acid sequences of carboxylesterases isolated from prepupal flea
larvae.
[0197] A. Anion Exchange Chromatography Fractions.
[0198] Anion exchange chromatography fractions described above in
Example 4 that contained proteins having CE activity were pooled,
diafiltered into TBS buffer and concentrated 3-fold in a Speed-Vac
Concentrator (available from Savant Instruments, Hoibrook, N.Y.).
Proteins in the concentrated samples were then resolved on a
reducing, 10% SDS-PAGE Tris-glycine gel (available from Novex) for
1 hour at about 200 V. The proteins on the gel were then blotted
onto a polyvinylidene difluoride (PVDF) membrane (available from
Novex) for about 70 min in 10 mM CAPS buffer
(3[cyclohexylamino]-1-propanesulfonic acid; available from Sigma),
pH 11, with 0.5 mM dithiothreitol (DTT). The membrane was then
stained for 1 minute in 0.1% Coomassie Blue R-250 dissolved in 40%
methanol and 1% acetic acid. The membrane was destained in 50%
methanol for about 10 minutes, rinsed with MilliQ water and air
dried. Three stained protein bands were identified having apparent
molecular weights of about 64 kD, 65 kD, and 66 kD, respectively.
The portion of the membrane containing each band was excised
separately. Protein contained in each membrane segment was
subjected to N-terminal amino acid sequencing using a 473A Protein
Sequencer (available from Applied Biosystems, Foster City, Calif.)
and using standard techniques.
[0199] The results indicated that the N-terminal amino acid
sequence of the putative 64 kD protein was DPPTVTLPQGEL (denoted
SEQ ID NO:39); the N-terminal amino acid sequence of the putative
65 kD protein was DPPTVTLPQGELVGKATNEnxk (denoted SEQ ID NO:40);
and the N-terminal amino acid sequence of the putative 66 kD
protein was DppTVTLPQGEL (denoted SEQ ID NO:41), in which the lower
case letters designate uncertainties and "x" designates an
undetermined residue.
[0200] B. Proteins Resolved by Native IEF-PAGE.
[0201] Proteins isolated by anion exchange chromatography as
described above in Section A were further resolved by native
IEF-PAGE. Proteins were loaded onto a pH 3-10 IEF gel (available
from Novex) and separated in Novex's IEF buffers according to
Novex's standard procedure (60 min at 100 V; then 60 min at 200 V;
and then 30 min at 500 V). Following electrophoresis, part of the
gel was stained for CE activity using the method described above in
Example 2. The remaining portion of the gel was blotted onto PVDF
membrane by reversing the orientation of the gel and membrane so
that positively charged proteins migrated to the membrane,
electrophoresing the protein for 60 min at 10 V, using 0.7% acetic
acid as the transfer buffer. The membrane was stained as described
above in Section A. After the membrane was dried, stained protein
bands on the membrane were compared to bands on the gel tested for
CE activity to identify corresponding bands. Protein bands on the
membrane corresponding to proteins having CE activity were excised
and submitted to N-terminal sequencing as described in Section
A.
[0202] N-terminal amino acid sequence was obtained for protein
contained in two bands having pI values of about pI 4.8 and about
pI 4.9. N-terminal amino acid sequence of the pI 4.8 band was
DPPTVTLPQGELVGKALSNen (denoted SEQ ID NO:42) and N-terminal amino
acid sequence of the pI 4.9 band was DPPTVTLP (denoted SEQ ID
NO:43). A comparison of the N-terminal amino acid sequences
identified here and described in Section A indicates closely
related proteins having a consensus sequence of
DPPTVTLPQGELVGKALTNEnGk (denoted SEQ ID NO:44).
[0203] The amino acid sequences of SEQ ID NO:39, SEQ ID NO:40, SEQ
ID NO:41, SEQ ID NO:42, SEQ ID NO:43 and SEQ ID NO:44 are
substantially contained within SEQ ID NO:5, SEQ ID NO:19 and SEQ ID
NO:53, which are described below in Example 11.
Example 6
[0204] This example describes partial purification of CE from 3rd
instar flea larvae.
[0205] Using the extract preparation methods described in Example 1
without labelling, extracts were prepared from about 50,000 bovine
blood-fed 3rd instar flea larvae. The extract was then further
purified over a p-aminobenzamidine linked agarose bead column using
the method also described in Example 1. Collected unbound protein
was concentrated to about 70 ml using a 200 ml stirred cell fitted
with a YM-10 membrane (available from Amicon, Beverly, Mass.).
Seven ml (about 5,000 3rd instar flea larval equivalents) of the
concentrated extract was used for the remainder of the purification
scheme described in Example 4. Resulting fractions from the anion
exchange chromatography column were tested for CE activity using
the assay described above in Example 2.
[0206] The results indicated that CE activity was eluted in two
overlapping peaks at about 120 mM and about 210 mM NaCl.
Example 7
[0207] This example describes the identification of JHE activity in
different flea tissues.
[0208] Tissue samples were prepared as described above in Example 1
from unfed and bovine blood-fed 1st instar flea larvae, bovine
blood-fed 3rd instar flea larvae, bovine blood-fed prepupal flea
larvae, unfed and cat blood-fed whole adult fleas, cat blood-fed
adult partial fleas and cat blood-fed adult flea midguts. About 5
tissue equivalents of each tissue was assayed for JHE activity as
follows.
[0209] Unlabeled juvenile hormone (JH; available from ICN
Biomedicals, Inc., Aurora, Ohio) was diluted in hexane to
concentration of about 0.025 M. Labeled 10.sup.-3H-juvenile hormone
(.sup.3H-JH; available from Dupont-NEN) was diluted in hexane to
concentration of about 80,000 cpm/.mu.l. A JH substrate mixture was
prepared by mixing about 20 .mu.l of unlabeled JH with about 80
.mu.l of .sup.3H-JH (about 5 .mu.Ci) in a 4 ml screw cap vial. The
substrate mixture was then covered with nitrogen (i.e.,
"blanketed") and the solvent contained in the mixture was
evaporated by heating the mixture at 35.degree. C. When just dry,
about 1 ml of absolute anhydrous ethanol (final concentration
5.times.10-4 M, or 6400 cpn/.mu.l) was added to the vial. The
substrate mixture was then stored at -200.degree. C.
[0210] About 5 equivalents of each tissue (about 5 .mu.l of
protein) was added into the bottom of a small glass autosampler
vial. About 95 .mu.l of Tris-buffered saline (TBS) was added to
each vial to bring the final volume in each vial to about 100
.mu.l. Two control samples were also prepared by adding 100 .mu.l
TBS to two separate vials. About 1 .mu.l of the substrate mixture
described above was added to all of the vials including the control
samples. The final JH concentration in each vial was about
5.times.10.sup.-6 M. The vials were then capped and spun in a
microfuge to bring all of the liquid to the bottom of each vial.
The vials were then transferred to a heat block and incubated at
35.degree. C. for about 30 minutes. Following the incubation,
enzyme activity was stopped by adding about 50 .mu.l of methanol
buffer (methanol:water:concentrated ammonium hydroxide at a 10:9:1
ratio, respectively) to each vial and removing the vials from the
heat block.
[0211] To measure labeled juvenile hormone acid, about 250 .mu.l
isooctane was added to each vial. Each vial was vortexed for about
15 seconds or until an emulsion formed. Each vial was then
centrifuged in a microfuge for about 1 minute to separate aqueous
and organic phases. About 75 .mu.l of the aqueous layer was removed
from each vial and added to about 2 nl Eco-1-me scintillation fluid
(available from ICN). The amount of .sup.3H-juvenile hormone acid
contained in each vial was determined using a Beckman LS-1801
liquid scintillation counter (available from Beckman, Fullerton,
Calif.).
[0212] The results shown in FIG. 5 indicated that all flea tissues
tested contain active JHE. Referring to Example 2, the level of CE
activity differed from JHE activity in various tissue samples. The
combined JHE and CE data indicated the differential expression of
these two enzymatic activities during the development of a
flea.
Example 8
[0213] This example describes the purification of JHE protein from
cat blood-fed adult midguts.
[0214] About 23,000 cat blood-fed adult midguts were collected and
prepared using the method described in Example 1. The extract was
then added in 4 aliquots to columns containing about 3 to about 5
ml of p-aminobenzamidine linked agarose beads (available from
Sigma), equilibrated in 50 mM Tris (pH 8.0), 100 mM CaCl.sub.2, 400
mM NaCl, and incubated overnight at 4.degree. C. The columns were
then washed with about 15 to about 125 ml of the equilibration Tris
buffer to remove unbound protein. The collected unbound protein was
pooled and then concentrated to a volume of about 5 ml using an
Ultrafree-20 10 kD centrifugal concentrator (available from
Millipore, Bedford, Mass.) and filtered sequentially through a 0.2
.mu.m centrifugal ultrafiltration membrane (available from Lida,
Kenosha, Wis.) to clarify the sample for chromatography.
[0215] Aliquots of about 0.5 ml were loaded onto a Superdex 200 HR
gel filtration column using the method described in Example 4.
Repeated runs were performed until about 10 ml of each fraction was
collected. The fractions were analyzed for JHE activity using the
assay described in Example 7. In preparation for anion exchange
chromatography, fractions having JHE activity (V.sub.e=17-18 ml)
were combined and dialyzed overnight against about 1 L of 20 mM
Tris buffer, pH 8.0, containing 10 mM NaCl. The sample was then
loaded onto a Poros 10 HQ anion exchange column using the method
described in Example 4. Resulting fractions were tested for JHE
activity as described in Example 7.
[0216] The results indicated that midgut JHE activity was eluted
from the anion exchange column in a single peak at about 120 mM
NaCl.
Example 9
[0217] This example describes partial purification of JHE from
prepupal flea larvae and 3rd instar larvae.
[0218] A. JHE Purification from Prepupal Tissue.
[0219] Using the extract preparation methods described in Example
1, gel filtration fractions were obtained using a Superdex 200 HR
gel filtration column (available from Pharmacia) using the method
described in Example 4, from about 15,000 bovine blood-fed prepupal
flea larvae. The fractions were analyzed for JHE activity using the
assay described above in Example 7. Those fractions containing
protein having JHE activity (V.sub.e=16-18 ml) were combined and
dialyzed using the method described in Example 8.
[0220] The fractions were then further purified by passing the
fractions over a Bio-Scale S2 cation exchange column (available
from BioRad) at a rate of about 0.5 ml/min. The column was washed
with MES until all unbound protein was eluted. Bound protein was
then eluted with a linear gradient of 20 mM MES buffer, pH 6.0,
containing 10 mM NaCl to 1 M NaCl. Resulting fractions were assayed
for JHE activity using the method described in Example 7. The
results indicated that proteins having JHE activity using prepupal
tissue eluted from the column in about 200 to 300 mM NaCl.
[0221] The fractions containing JHE activity were combined and the
pH adjusted to pH 7 using 0.5 M Tris buffer (pH 8.0). The fractions
were then dialyzed twice against about 1 liter of 10 mM phosphate
buffer (pH 7.2) containing 10 mM NaCl at 4.degree. C. The resulting
dialyzed fractions were then loaded onto a Bio-Scale CHT2-I
Hydroxyapatite Column (available from BioRad) at a rate of about
0.5 ml/min. Unbound protein was washed from the column using the
dialysis buffer. Bound protein was then eluted with a linear
gradient of from 10 mM phosphate buffer, pH 7.2, containing 10 mM
NaCl to 0.5 M phosphate buffer pH 6.5 containing 10 mM NaCl. One ml
fractions were collected and each tested for JHE activity by the
method described in Example 7.
[0222] The results indicated that JHE eluted in 2 overlapping peaks
at about 100 mM and 150 mM phosphate. These two JHE activities were
designated PF JHEI and PP JHE II, and were kept separate for the
remainder of the purification. Both JHE samples were dialyzed
overnight against 20 mM Tris buffer (pH 8.0) containing 10 mM NaCl.
The two samples were then loaded, separately, onto a 4.5
mm.times.50 mm Poros 10 HQ anion exchange chromatography column
(available from PerSeptive Biosystems) equilibrated with 20 mM Tris
buffer, pH 8.0, containing 10 mM NaCl. Unbound proteins were washed
from the column using the same buffer. Bound proteins were eluted
with a linear gradient of from 10 mM to 1 M NaCl in 20 mM Tris
buffer, pH 8.0. Resulting fractions were tested for JHE activity
using the method described in Example 7.
[0223] The results indicated that in both samples, JHE activity was
eluted from the column in a single peak at about 100 mM NaCl.
[0224] B. JHE Purification from 3.sup.rd Instar Tissue
[0225] Using the procedure described above in Section A, proteins
having JHE activity were obtained using about 5,000 bovine
blood-fed 3.sup.rd instar flea larvae. Following purification by
cation exchange, proteins having JHE activity using 3.sup.rd instar
tissue were found to elute in 2 peaks. The first peak having JHE
activity was not retained on the column and also exhibited CE
activity (referred to herein as CE/JHE fractions). The second peak
having JHE activity eluted from the column in about 100-200 mM NaCl
and did not contain CE activity.
[0226] The CE/JHE fractions were pooled and adjusted to about pH 7
using 0.5 M Tris, pH 8.0. The fractions were then loaded onto a 4.5
mm.times.50 mm Poros 10 HQ anion exchange chromatography column
(available from PerSeptive Biosystems) and the column was
equilibrated in 25 mM Tris buffer, pH 6.8. The column was washed
with the same buffer and bound proteins were eluted with a linear
gradient of 0 to 1 M NaCl in 25 mM Tris buffer, pH 6.8. Fractions
were then tested for JHE activity using the method described in
Example 7. JHE activity was eluted in two overlapping peaks at
about 120 mM and 210 mM NaCl. The fraction containing JHE activity
also contained CE activity when tested using the method described
in Example 2.
[0227] Fractions from the cation exchange column containing only
JHE activity were combined, diluted in 20 mM Tris buffet, pH 8.0
containing 10 mM NaCl, and concentrated to about 5 ml. The
fractions were purified on a Poros 10 HQ anion exchange
chromatography column as described immediately above. Fractions
were then tested for JHE activity using the method described in
Example 7. The JHE activity was eluted in a single peak at about
120 mM. The peak contained no detectable CE activity.
Example 10
[0228] This example describes the purification of JHE protein from
unfed adult midguts.
[0229] About 16,000 unfed adult midguts were collected in 20 mM
Tris buffer (pH 7.7), containing 130 mM NaCl, 1 mM sodium EDTA, 1
mM Pefabloc.RTM. (available from Boehringer Mannheim, Indianapolis,
Ind.), 1 microgram/ml (.mu.g/ml) leupeptin and 1 .mu.g/ml
pepstatin. The midguts were homogenized by freeze-fracture and
sonication, and then centrifuged at about 14,000.times.g for 20
min. The soluble material from the centrifugation step was
recovered. The soluble material was then concentrated to about 1 ml
using an Ultrafree-20 10 kD centrifugal concentrator (available
from Millipore) and filtered sequentially through a 0.2 .mu.m
centrifugal ultrafiltration membrane to clarify the sample for
chromatography. Aliquots of about 0.5 ml were loaded onto a
Superdex 200 HR gel filtration column using the method described in
Example 4. Repeated column runs were performed until about 2 ml of
each fraction was collected. The fractions were analyzed for JHE
activity using the assay described in Example 7. In preparation for
cation exchange chromatography, fractions having JHE activity
(V.sub.e=15-17 ml) were combined and dialyzed overnight against
about 1 L of 20 mM MES buffer, pH 6.0, containing 10 mM NaCl. The
sample was then applied to a Bio-Scale S2 cation exchange column
using the method described in Example 4. Fractions of eluate were
assayed for JHE activity using the method described in Example
7.
[0230] The results indicate that JHE is present in unfed midguts in
two forms, one that is not retained on the cation exchange column
and one that is bound to the column under low salt conditions at
about 100 mM NaCl. The form that was not retained under low salt
conditions was shown to have general CE activity using the methods
described in Example 2.
Example 11
[0231] This example describes the identification of certain
esterase nucleic acid molecules of the present invention.
[0232] Several flea esterase nucleic acid molecules, representing
one or more partial flea esterase genes, were PCR amplified from a
flea mixed instar cDNA library or a flea prepupal cDNA library. The
flea mixed instar cDNA library was produced using unfed 1 st
instar, bovine blood-fed 1 st instar, bovine blood-fed 2.sup.nd
instar and bovine blood-fed 3.sup.rd instar flea larvae (this
combination of tissues is referred to herein as mixed instar larval
tissues for purposes of this example). The flea prepupal cDNA
library was produced using prepupal flea larvae. For each library,
total RNA was extracted from mixed instar or prepupal tissue,
respectfully, using an acid-guanidinium-phenol-chlorofor- m method
similar to that described by Chomczynski et al., 1987, Anal.
Biochem. 162, p. 156-159. Approximately 5,164 mixed instar larvae
or 3,653 prepupal larvae were used in each RNA preparation. Poly A+
selected RNA was separated from each total RNA preparation by
oligo-dT cellulose chromatography using Poly(A)Quick.RTM. mRNA
isolation kits (available from Stratagene Cloning Systems, La
Jolla, Calif.), according to the method recommended by the
manufacturer.
[0233] A mixed instar cDNA expression library and a prepupal cDNA
expression library were constructed in lambda (.lambda.)
Uni-ZAP.TM.XR vector (available from Stratagene Cloning Systems)
using Stratagene's ZAP-cDNA Synthesis Kit.RTM. protocol. About 6.34
.mu.g of mixed instar poly A+ RNA were used to produce the mixed
instar library and about 6.72 .mu.g of prepupal poly A+ RNA were
used to produce the prepupal library. The resultant mixed instar
library was amplified to a titer of about 2.17.times.1010 pfu/ml
with about 97% recombinants. The resultant prepupal library was
amplified to a titer of about 3.5.times.10.sup.10 pfu/ml with about
97% recombinants.
[0234] A pair of primers was used to amplify DNA from the cDNA
libraries. A sense vector primer T-3.times. (corresponding to the
vector in which nucleic acid molecules of the present invention had
been ligated), having the nucleic acid sequence AATTAACCCT
CACTAAAGGG (available from Gibco BRL, Gaithersburg, Md.; denoted
SEQ ID NO:45), was used in combination with a degenerate primer,
the design of which was based on a highly conserved esterase amino
acid sequence (disclosed in Hanzlik et al., J. Biol. Chem.
264:12419-12423, 1989; I Y/H G G G F/L) located in a region
downstream from the mature amino terminus in a number of known
esterases. The degenerate primer, referred to herein as FCEF, is an
anti-sense primer having the nucleic acid sequence ARDCCDCCDC
CRTRDAT (R indicating an A or G; and D indicating an A, G or T;
denoted SEQ ID NO:46). The resultant PCR products from the mixed
instar cDNA library, obtained using standard PCR conditions (e.g.,
Sambrook et al., ibid.), were about 550 nucleotides. The resultant
PCR products from the prepupal cDNA library were from about 500
nucleotides to about 860 nucleotides.
[0235] A. PCR Products.
[0236] PCR products were gel purified and cloned into the TA
Vector.TM. (available from InVitrogen Corp., San Diego, Calif.).
Approximately 8 clones were identified from the prepupal library
and 6 clones were identified from the mixed instar library. These
nucleic acid molecules were subjected to nucleic acid sequencing
using the Sanger dideoxy chain termination method, as described in
Sambrook et al., ibid.
[0237] 1. Flea esterase clone 1 isolated from the mixed instar cDNA
library was determined to comprise nucleic acid molecule
nfE1.sub.401, the nucleic acid sequence of the coding strand which
is denoted herein as SEQ ID NO:1. Translation of SEQ ID NO:1
suggests that nucleic acid molecule nfE1.sub.401, encodes a
non-full-length flea esterase protein of about 103 amino acids,
referred to herein as PfE1.sub.103, having amino acid sequence SEQ
ID NO:2, assuming an initiation codon spanning from nucleotide 92
through nucleotide 94 of SEQ ID NO:1. The complement of SEQ ID NO:1
is represented herein by SEQ ID NO:3. Comparison of amino acid
sequence SEQ ID NO:2 (i.e., the amino acid sequence of
PfE1.sub.103) with amino acid sequences reported in GenBank
indicates that SEQ ID NO:2, showed the most homology, i.e., about
33% identity, between SEQ ID NO:2 and alpha esterase protein from
Drosophila melanogaster.
[0238] 2. Flea esterase clone 2 isolated from the mixed instar cDNA
library was determined to comprise nucleic acid molecule
nfE2.sub.364, the nucleic acid sequence of the coding strand which
is denoted herein as SEQ ID NO:4. Translation of SEQ ID NO:4
suggests that nucleic acid molecule nfE2.sub.364 encodes a
non-full-length flea esterase protein of about 121 amino acids,
referred to herein as PfE2.sub.121, having amino acid sequence SEQ
ID NO:5, assuming the first codon spans from nucleotide 2 through
nucleotide 4 of SEQ ID NO:4. The complement of SEQ ID NO:4 is
represented herein by SEQ ID NO:6. Comparison of nucleic acid
sequence SEQ ID NO:4 with nucleic acid sequences reported in
GenBank indicates that SEQ ID NO:4 showed the most homology, i.e.,
about 43% identity, between SEQ ID NO:4 and a H. virescens JHE
gene. Comparison of amino acid sequence SEQ ID NO:5 (i.e., the
amino acid sequence of PfE2.sub.121) with amino acid sequences
reported in GenBank indicates that SEQ ID NO:5, showed the most
homology, i.e., about 38% identity, between SEQ ID NO:5 and alpha
esterase protein from Drosophila melanogaster.
[0239] 3. Flea esterase clone 3 isolated from the prepupal cDNA
library was determined to comprise nucleic acid molecule
nfE3.sub.421, the nucleic acid sequence of the coding strand which
is denoted herein as SEQ ID NO:7. Translation of SEQ ID NO:7
suggests that nucleic acid molecule nfE3.sub.421 encodes a
non-full-length flea esterase protein of about 103 amino acids,
referred to herein as PfE3.sub.103, having amino acid sequence SEQ
ID NO:8, assuming an initiation codon spanning from nucleotide 113
through nucleotide 115 of SEQ ID NO:7. The complement of SEQ ID
NO:7 is represented herein by SEQ ID NO:9. Comparison of nucleic
acid sequence SEQ ID NO:7 with nucleic acid sequences reported in
GenBank indicates that SEQ ID NO:7 showed the most homology, i.e.,
about 53% identity, between SEQ ID NO:7 and a Torpedo marmorata
acetylcholinesterase gene. Comparison of amino acid sequence SEQ ID
NO:8 (i.e., the amino acid sequence of PfE3.sub.103) with amino
acid sequences reported in GenBank indicates that SEQ ID NO:8,
showed the most homology, i.e., about 39% identity, between SEQ ID
NO:5 and alpha esterase protein from Drosophila melanogaster.
[0240] 4. Flea esterase clone 4 isolated from the prepupal cDNA
library was determined to comprise nucleic acid molecule
nfE4.sub.524, the nucleic acid sequence of the coding strand which
is denoted herein as SEQ ID NO:10. Translation of SEQ ID NO:10
suggests that nucleic acid molecule nfE4.sub.524 encodes a
non-full-length flea esterase protein of about 137 amino acids,
referred to herein as PfE4.sub.137, having amino acid sequence SEQ
ID NO:11, assuming an initiation codon spanning from nucleotide 113
through nucleotide 115 of SEQ ID NO:10. The complement of SEQ ID
NO:10 is represented herein by SEQ ID NO:12. Comparison of nucleic
acid sequence SEQ ID NO:10 with nucleic acid sequences reported in
GenBank indicates that SEQ ID NO:10 showed the most homology, i.e.,
about 47% identity, between SEQ ID NO:10 and an Anas platyrhyncos
thioesterase B gene. Comparison of amino acid sequence SEQ ID NO:11
(i.e., the amino acid sequence of PfE4.sub.137) with amino acid
sequences reported in GenBank indicates that SEQ ID NO:11, showed
the most homology, i.e., about 30% identity, between SEQ ID NO:11
and Leptinotarsa decemlineata acetylcholinesterase.
[0241] B. cDNA Clones.
[0242] Certain amplified PCR fragments were used as probes to
identify full-length flea esterase genes in the prepupal cDNA
library.
[0243] 1. Nucleic acid molecule nfE2.sub.364 was labeled with
.sup.32P and used as a probe to screen the mixed instar cDNA
library described in Section A, using standard hybridization
techniques. Two clones were isolated. A first clone included about
a 2300-nucleotide insert, referred to herein as nfE5.sub.2300.
Nucleic acid sequence was obtained using standard techniques from
nfE5.sub.2300, to yield a flea esterase nucleic acid molecule named
nfE5.sub.982 having a nucleic acid sequence of the coding strand
which is denoted herein as SEQ ID NO:13. Translation of SEQ ID
NO:13 suggests that nucleic acid molecule nfE5 .sub.1982 encodes a
non-full-length flea esterase protein of about 505 amino acids,
referred to herein as PfE5.sub.505, having amino acid sequence SEQ
ID NO:14, assuming the first codon spans from nucleotide 1 through
nucleotide 3 of SEQ ID NO:13 and the stop codon spans from
nucleotide 1518 through nucleotide 1520 of SEQ ID NO:13. The
complement of SEQ ID NO:13 is represented herein by SEQ ID NO:15.
The amino acid sequence of PfE5.sub.505 (i.e., SEQ ID NO:14)
predicts that PfE5.sub.505 has an estimated molecular weight of
about 56.8 kD and an estimated pI of about 5.5. The nucleic acid
molecule representing the coding region for PfE5.sub.505 is
referred to herein as nfE5.sub.1515; the nucleic acid sequences of
the coding strand and the complementary strand are represented by
SEQ ID NO:16 and SEQ ID NO:17, respectively.
[0244] The nucleic acid sequence of nfE5.sub.1982 was used to
design primers to use in combination with a vector primer to PCR
amplify the 5' terminal fragment of the remainder of the flea
esterase coding region from the flea mixed instar cDNA library. A
pair of primers was used to amplify DNA from the cDNA library. A
sense vector primer T3-X (corresponding to the vector in which
nucleic acid molecules of the present invention had been ligated),
having the nucleic acid sequence 5' AATTAACCCT CACTAAAGCG 3'
(denoted SEQ ID NO:45), was used in combination with an anti-sense
primer M6/M265', having the nucleic acid sequence 5' GTGCGTACAC
GTTTACTACC 3' (denoted SEQ ID NO:56). The resultant PCR product
from the mixed instar cDNA library, obtained using standard PCR
conditions (e.g., Sambrook et al., ibid.), were about 354
nucleotides.
[0245] The PCR product was subjected to DNA sequencing analysis,
and a composite sequence representing a full-length flea esterase
coding region was deduced. The nucleic acid sequence of the
composite nucleic acid molecule, referred to herein as
nfE5.sub.2144 is denoted herein as SEQ ID NO:57. Translation of SEQ
ID NO:57 suggests that nucleic acid molecule nfE5.sub.2144 encodes
a full-length flea esterase protein of about 550 amino acids,
referred to herein as PfE5.sub.550, having amino acid sequence SEQ
ID NO:58, assuming an open reading frame in which the initiation
codon spans from nucleotide 30 through nucleotide 32 of SEQ ID
NO:57 and the stop codon spans from nucleotide 1680 through
nucleotide 1682 of SEQ ID NO:57. The complement of SEQ ID NO:57 is
represented herein by SEQ ID NO:59. The coding region encoding
PfE5.sub.550 is represented by the nucleic acid molecule
nfE5.sub.1650, having a coding strand with the nucleic acid
sequence represented by SEQ ID NO:60 and a complementary strand
with nucleic acid sequence SEQ ID NO:61. The amino acid sequence of
PfE5.sub.550 (i.e., SEQ ID NO:58) predicts that PfE5.sub.550 has an
estimated molecular weight of about 61.8 kD and an estimated pI of
about 5.5.
[0246] Comparison of nucleic acid sequence SEQ ID NO:57 with
nucleic acid sequences reported in GenBank indicates that SEQ ID
NO:57 showed the most homology, i.e., about 41% identity, between
SEQ ID NO:57 and a M. persicae esterase FE4 mRNA sequence.
Comparison of amino acid sequence SEQ ID NO:58 (i.e., the amino
acid sequence of PfE5.sub.550) with amino acid sequences reported
in GenBank indicates that SEQ ID NO:58 showed the most homology,
i.e., about 36% identity between SEQ ID NO:58 and Drosophila
melanogster alpha esterase protein.
[0247] A second clone included about a 1900 nucleotide insert,
referred to herein as nfE6.sub.1900. Nucleic acid sequence was
obtained using standard techniques from nfE6.sub.1900, to yield a
flea esterase nucleic acid molecule named nfE6.sub.1792 having a
nucleic acid sequence of the coding strand which is denoted herein
as SEQ ID NO:18. Translation of SEQ ID NO:18 suggests that nucleic
acid molecule nfE6.sub.1792 encodes a full-length flea esterase
protein of about 550 amino acids, referred to herein as
PfE6.sub.550, having amino acid sequence SEQ ID NO:19, assuming an
open reading frame in which the initiation codon spans from
nucleotide 49 through nucleotide 51 of SEQ ID NO:18 and a stop
codon spanning from nucleotide 1699 through nucleotide 1701 of SEQ
ID NO:18. The complement of SEQ ID NO:18 is represented herein by
SEQ ID NO:20. The coding region encoding PfE6.sub.550, is
represented by nucleic acid molecule nfE6.sub.1650, having a coding
strand with the nucleic acid sequence represented by SEQ ID NO:21
and a complementary strand with nucleic acid sequence SEQ ID NO:22.
The proposed mature protein, denoted herein as PfE6.sub.530,
contains about 530 amino acids which is represented herein as SEQ
ID NO:53. The nucleic acid molecule encoding PfE6.sub.530 is
denoted herein as nfE6.sub.590 and has a coding strand having the
nucleic acid sequence SEQ ID NO:23. The amino acid sequence of
PfE6.sub.550 (i.e., SEQ ID NO:19) predicts that PfE6.sub.550 has an
estimated molecular weight of about 61.8 kD and an estimated pI of
about 5.5. Comparison of nucleic acid sequence SEQ ID NO:18 with
nucleic acid sequences reported in GenBank indicates that SEQ ID
NO:18 showed the most homology, i.e., about 41% identity, between
SEQ ID NO:18 and a Myzus pericae esterase gene. Comparison of amino
acid sequence SEQ ID NO:19 (i.e., the amino acid sequence of
PfE6.sub.550) with amino acid sequences reported in GenBank
indicates that SEQ ID NO:19 showed the most homology, i.e., about
28% identity between SEQ ID NO:19 and Drosophila melanogaster alpha
esterase protein.
[0248] 2. Nucleic acid molecule nfE4.sub.524 was labeled with
.sup.32P and used as a probe to screen the prepupal cDNA library
described in Example 11, using standard hybridization techniques
(e.g., Sambrook et al., ibid.). Two clones were isolated. A first
clone included about a 3000 nucleotide insert, referred to herein
as nfE7.sub.3000. Nucleic acid sequence was obtained using standard
techniques from nfE7.sub.3000, to yield a flea esterase nucleic
acid molecule named nfE7.sub.2836 having a nucleic acid sequence of
the coding strand which is denoted herein as SEQ ID NO:24.
Translation of SEQ ID NO:24 suggests that nucleic acid molecule
nfE7.sub.236 encodes a full-length flea esterase protein of about
596 amino acids, referred to herein as PfE7.sub.596, having amino
acid sequence SEQ ID NO:25, assuming an open reading frame in which
the initiation codon spans from nucleotide 99 through nucleotide
101 of SEQ ID NO:24 and a stop codon spanning from nucleotide 1887
through nucleotide 1889 of SEQ ID NO:25. The complement of SEQ ID
NO:24 is represented herein by SEQ ID NO:26. The coding region
encoding PfE7.sub.596, is represented by nucleic acid molecule
nfE7.sub.1788, having a coding strand with the nucleic acid
sequence represented by SEQ ID NO:28 and a complementary strand
with nucleic acid sequence SEQ ID NO:29. The proposed mature
protein, denoted herein as PfE7.sub.570, contains about 570 amino
acids which is represented herein as SEQ ID NO:54. The nucleic acid
molecule encoding PfE7.sub.570 is denoted herein as nfE7.sub.1710
and has a coding strand having the nucleic acid sequence SEQ ID
NO:27. The amino acid sequence of PfE7.sub.596 (i.e., SEQ ID NO:25)
predicts that PfE7.sub.596 has an estimated molecular weight of
about 68.7 kD and an estimated pI of about 6.1.
[0249] Comparison of nucleic acid sequence SEQ ID NO:24 with
nucleic acid sequences reported in GenBank indicates that SEQ ID
NO:24 showed the most homology, i.e., about 48% identity, between
SEQ ID NO:24 and an Anas platychyncos thioesterase B gene.
Comparison of amino acid sequence SEQ ID NO:25 (i.e., the amino
acid sequence of PfE7.sub.596) with amino acid sequences reported
in GenBank indicates that SEQ ID NO:25 showed the most homology,
i.e., about 27% identity between SEQ ID NO:25 and Drosophila
melanogaster, alpha esterase protein.
[0250] A second clone included about a 3000 nucleotide insert,
referred to herein as nfE8.sub.3000. Nucleic acid sequence was
obtained using standard techniques from nfE8.sub.3000, to yield a
flea esterase nucleic acid molecule named nfE8.sub.280, having a
nucleic acid sequence of the coding strand which is denoted herein
as SEQ ID NO:30. Translation of SEQ ID NO:30 suggests that nucleic
acid molecule nfE8.sub.280, encodes a full-length flea esterase
protein of about 59: amino acids, referred to herein as
PfE8.sub.595, having amino acid sequence SEQ ID NO:31, assuming an
open reading frame in which the initiation codon spans from
nucleotide 99 through nucleotide 101 of SEQ ID NO:30 and a stop
codon spanning from nucleotide 1884 through nucleotide 1886 of SEQ
ID NO:30. The complement of SEQ ID NO:30 is represented herein by
SEQ ID NO:32. The coding region encoding PfE8.sub.595, is
represented by nucleic acid molecule nfE8.sub.1785, having a coding
strand with the nucleic acid sequence represented by SEQ ID NO:34
and a complementary strand with nucleic acid sequence SEQ ID NO:35.
The proposed mature protein, denoted herein as PfE8.sub.570,
contains about 570 amino acids which is represented herein as SEQ
ID NO:55. The nucleic acid molecule encoding PfE8.sub.570 is
denoted herein as nfE8.sub.1710 and has a coding strand having the
nucleic acid sequence SEQ ID NO:33. The amino acid sequence of
PfE8.sub.595 (i.e., SEQ ID NO:31) predicts that PfE8.sub.595 has an
estimated molecular weight of about 68.6 kD and an estimated pI of
about 6.1.
[0251] Comparison of nucleic acid sequence SEQ ID NO:30 with
nucleic acid sequences reported in GenBank indicates that SEQ ID
NO:30 showed the most homology, i.e., about 46% identity, between
SEQ ID NO:30 and a Mus musculus carboxyl ester lipase gene.
Comparison of amino acid sequence SEQ ID NO:31 (i.e., the amino
acid sequence of PfE8.sub.595) with amino acid sequences reported
in GenBank indicates that SEQ ID NO:31 showed the most homology,
i.e., about 28% identity between SEQ ID NO:31 and estalpha-2
esterase of Culex pipens quinque fasciatus.
[0252] 3. Nucleic acid molecule nfE3.sub.421 was labeled with
.sup.32P and used as a probe to screen the prepupal cDNA library
using standard hybridization techniques (e.g., Sambrook et al.,
ibid.). Two clones were isolated. One clone included about a 1900
nucleotide insert, referred to herein as nfE9.sub.1900. Nucleic
acid sequence was obtained using standard techniques from
nfE9.sub.1900, to yield a flea esterase nucleic acid molecule named
nfE9.sub.2007 having nucleic acid sequence of the coding strand
which is denoted herein as SEQ ID NO:36. Translation of SEQ ID
NO:36 suggests that nucleic acid molecule nfE9.sub.2007 encodes a
full-length flea esterase protein of about 528 amino acids,
referred to herein as PfE9.sub.528, having amino acid sequence SEQ
ID NO:37, assuming an open reading frame in which the initiation
codon spans from nucleotide 11 through nucleotide 13 of SEQ ID
NO:36 and a stop codon spanning from nucleotide 1595 through
nucleotide 1597 of SEQ ID NO:36. The complement of SEQ ID NO:36 is
represented herein by SEQ ID NO:38. The coding region encoding
PfE9.sub.528, is represented by nucleic acid molecule
nfE9.sub.1584, having a coding strand with the nucleic acid
sequence represented by SEQ ID NO:51 and a complementary strand
with nucleic acid sequence SEQ ID NO:52. The amino acid sequence of
PfE9.sub.528 (i.e., SEQ ID NO:37) predicts that PfE9.sub.528 has an
estimated molecular weight of about 60 kD and an estimated pI of
about 5.43.
[0253] Comparison of nucleic acid sequence SEQ ID NO:36 with
nucleic acid sequences reported in GenBank indicates that SEQ ID
NO:36 showed the most homology, i.e., about 47% identity, between
SEQ ID NO:36 and a hamster mRNA for carboxylesterase precursor
gene. Comparison of amino acid sequence SEQ ID NO:37 (i.e., the
amino acid sequence of PfE9.sub.528) with amino acid sequences
reported in GenBank indicates that SEQ ID NO:37 showed the most
homology, i.e., about 37% identity between SEQ ID NO:37 and alpha
esterase protein from Drosophila melanogaster.
[0254] As is the case for any of the nucleic acid molecules
described in this example, variations between sequences may be due
to a number of factors, such as but not limited to, sequencing
errors or allelic variation.
[0255] 4. Nucleic acid molecule nfE1.sub.401 was labeled with
.sup.32P and used as a probe to screen the mixed instar cDNA
library using standard hybridization techniques (e.g., Sambrook et
al., ibid.). A clone was isolated that included about a 2000
nucleotide insert, referred to herein as nfE10.sub.2000. Nucleic
acid sequence was obtained using standard techniques from
nfE10.sub.2000, to yield a flea esterase nucleic acid molecule
named nfE10.sub.987 having nucleic acid sequence of the coding
strand which is denoted herein as SEQ ID NO:67. Translation of SEQ
ID NO:67 suggests that nucleic acid molecule nfE10.sub.987 encodes
a full-length flea esterase protein of about 530 amino acids,
referred to herein as PfE10.sub.530, having amino acid sequence SEQ
ID NO:68, assuming an open reading frame in which the initiation
codon spans from nucleotide 231 through nucleotide 233 of SEQ ID
NO:67 and a stop codon spanning from nucleotide 1821 through
nucleotide 1823 of SEQ ID NO:67. The complement of SEQ ID NO:67 is
represented herein by SEQ ID NO:69. The coding region encoding
PfE10.sub.530, is represented by nucleic acid molecule
nfE10.sub.590, having a coding strand with the nucleic acid
sequence represented by SEQ ID NO:70 and a complementary strand
with nucleic acid sequence SEQ ID NO:71. The amino acid sequence of
PfE10.sub.530(i.e., SEQ ID NO:68) predicts that PfE10.sub.530 has
an estimated molecular weight of about 59.5 kD and an estimated pI
of about 5.5.
[0256] Comparison of nucleic acid sequence SEQ ID NO:67 with
nucleic acid sequences reported in GenBank indicates that SEQ ID
NO:67 showed the most homology, i.e., about 48% identity, between
SEQ ID NO:67 and a Lucilia cuprila alpha esterase gene (genembl
#U56636) gene. Comparison of amino acid sequence SEQ ID NO:68
(i.e., the amino acid sequence of PfE10.sub.530) with amino acid
sequences reported in GenBank indicates that SEQ ID NO:68 showed
the most homology, i.e., about 30% identity between SEQ ID NO:68
and Culex pipens esterase b1 precurser protein (swissprot
#P16854).
[0257] As is the case for any of the nucleic acid molecules
described in this example, variations between sequences may be due
to a number of factors, such as but not limited to, sequencing
errors or allelic variation.
Example 12
[0258] This Example demonstrates the production of esterase
proteins of the present invention in E. coli cells.
[0259] A. Flea esterase protein PHIS-PfE7.sub.570 and flea esterase
protein PHIS-PfE8.sub.570 were produced in the following manner. A
pair of primers was used to amplify DNA from flea esterase nucleic
acid molecule nfE7.sub.2836 or nfE8.sub.2801 produced as described
in Example 11. A sense primer containing an XhoI site (shown in
bold) having the nucleic acid sequence 5' TGTGCTCGAG ATGGGATAAC
CTAGATCAGC ATTTGTGC 3' (denoted SEQ ID NO:47), was used in
combination with an anti-sense primer containing a KpnI site (shown
in bold) having the nucleic acid sequence 5' TTAAGGTACC TCATCTAATA
CTTCCTTCAT TACAG 3' (denoted SEQ ID NO:48). A PCR product was
derived from nfE7.sub.2836, and is referred to herein
nfE7.sub.1710, having nucleic acid sequence SEQ ID NO:27. The PCR
product was digested with XhoI and KpnI restriction endonucleases,
gel purified and subcloned into expression vector pTrcHisB
(available from InVitrogen). The resultant recombinant molecule,
referred to herein as pTrc-nfE7.sub.1710, was transformed into E.
coli HB101 competent cells (available from Gibco BRL) to form
recombinant cell E. coli:pTrc-nfE7.sub.1710.
[0260] The PCR product derived from nfE8.sub.2801 using the primers
is referred to herein as nfE8.sub.1710, having nucleic acid
sequence SEQ ID NO:33. PCR product nfE8.sub.1710 was digested with
XhoI and KpnI restriction endonucleases, gel purified and subcloned
into expression vector pTrcHisB. The resultant recombinant
molecule, referred to herein as pTrc-nfE8.sub.1710, was transformed
into E. coli HB101 competent cells to form recombinant cell E.
coli:pTrc-nfE8.sub.1710.
[0261] The recombinant cells were cultured in enriched bacterial
growth medium containing 0.1 mg/ml ampicillin and 0.1% glucose at
about 32.degree. C. When the cells reached an OD.sub.600 of about
0.4-0.5, expression of recombinant protein was induced by the
addition of 0.5 mM isopropyl-B-D-thiogalactoside (IPTG), and the
cells were cultured for about 2 hours at about 32.degree. C.
Immunoblot analysis of recombinant cell E. coli:pTrcnfE7.sub.1710
and E. coli:pTrc-nfE8.sub.1710 lysates using a T7 tag monoclonal
antibody (available from Novagen, Inc., Madison, Wis.) directed
against the fusion portion of the recombinant PHIS-PfE7.sub.570 and
PHIS-PfE8.sub.570 fusion proteins identified proteins of
appropriate size, namely an about 65 kD protein for each fusion
protein.
[0262] B. Flea esterase protein PHIS-PfE6.sub.540 was produced in
the following manner. A pair of primers was used to amplify DNA
from flea esterase nucleic acid molecule nfE6.sub.1792 produced as
described in Example 11. A sense primer containing an XhoI site
having the nucleic acid sequence 5' AAACTCGAGT CCCCCGACTG TAACTTTGC
3' (denoted SEQ ID NO:62; XhoI site shown in bold), was used in
combination with an anti-sense primer containing a PstI site having
the nucleic acid sequence 5' TCATCTGCAG TTATTGACTG TGCAAAGTTT
TTGTGG 3' (denoted SEQ ID NO:63; PstI site shown in bold). A PCR
product was derived from nfE6.sub.1792, and is referred to herein
as nfE6.sub.1488, having nucleic acid sequence SEQ ID NO:76. The
PCR product was digested with XhoI and PstI restriction
endonucleases, gel purified and subcloned into expression vector
lambdaP.sub.R/T.sup.2ori/S10HIS-RSET-A9, that had been digested
with XhoI and PstI and dephosphorylated. The resultant recombinant
molecule, referred to herein as pCro-nfE6.sub.1488, was transformed
into E. coli HB101 competent cells (available from Gibco BRL) to
form recombinant cell E. coli:pCro-nfE6.sub.1488.
[0263] The recombinant cells were cultured using the method
generally described in Section A of this example, except that the
cells were grown under heat shift conditions rather than in the
presence of IPTG. The cells were grown at 32.degree. C. for about 2
hours, and then grown at 42.degree. C. Immunoblot analysis of
recombinant cell E. coli:pCro-nfE6.sub.1488 lysate using a T7 tag
monoclonal antibody directed against the fusion portion of the
recombinant PHIS-PfE6.sub.540 fusion protein identified proteins of
appropriate size, namely an about 60 kD protein for each fusion
protein.
[0264] Expression of the recombinant PHIS-PfE6.sub.540 fusion
protein was improved by transforming supercoiled plasmid
pCro-nfE6.sub.1488 DNA harested from E. coli:pCronfE6.sub.1488
cells into the BL-21 strain of E. coli (available from Novagen).
The amount of expression PHIS-PfE6.sub.540 was confined by
immunoblot using the method described immediately above.
[0265] E. coli cells expressing PHIS-PfE6.sub.540 protein were
harvested from about 2 liters of media and suspended in about 140
ml of 50 mM Tris, pH 8.0, 50 mM NaCl, 0.1 mM
phenylmethylsulfonylfluoride (PMSF) (Solubilization Buffer). The
cells were broken by passage through a microfluidizer at 30 psi for
30 cycles. The sample was centrifuged at about 16,000.times.g for
30 min at 40.degree. C. The supernatant (S1) was recovered and the
pellet was resuspended in about 80 ml of Solubilization Buffer and
centrifuged at about 16,000.times. g for 30 min at 40.degree. C.
The supernatant (S2) was recovered and the pellet was resuspended
in about 80 ml of Solubilization Buffer containing 0.1% Triton-X100
and centrifuged at about 16,000.times. g for 30 min at 40.degree.
C. The supernatant (S3) was recovered and the pellet was
resuspended in about 140 mls 50 mM Tris, pH 8.0, 8 M Urea, 0.1 M
PMSF and centrifuged at about 16,000.times. g. The supernatant (S4)
was recovered and the pellet was resuspended in 40 mis 50 mM Tris,
8 M Urea, 0.1 M PMSF. Aliquots of each pellet and supernatant were
analyzed by SDS-PAGE and immunoblot using the T7 tag monoclonal
antibody described above. The results indicated that the
PHIS-PfE6.sub.540 protein was located in the final supernatant
(S4). The PHIS-PfE6.sub.540 protein was loaded onto a 5.0 ml, Metal
chelating HiTrap column charged with NiCl.sub.2 (obtained from
Pharmacia Biotech Inc., Piscataway, N.J.), previously equilibrated
with 50 mM Tris, 1 mM PMSF, 1 mM .beta.-mercaptoethanol (OME), 8 M
urea, pH 8.0 (Buffer A). The column was washed with 10 column
volumes (cv) of Buffer A and then with 10 cv with 50 mM Tris, 25 mM
sodium acetate, 1 mM PMSF, 1 mM .beta.ME, 8 M urea, pH 6.0 (Buffer
B) to remove loosely bound proteins. Bound PHIS-PfE6.sub.540
protein was eluted with 10 cv of 50 mM Tris, 25 mM sodium acetate,
1 mM PMSF, 1 mM .beta.ME, 8 M urea, pH 4.0 (Buffer C). Column
fractions were analyzed for the presence of PHIS-PfE6.sub.540
protein by immunoblot using the T7 tag monoclonal antibody as
described above. The results indicated that the majority of the
PHIS-PfE6.sub.540 protein was eluted by Buffer C. The fractions
containing the PHIS-PfE6.sub.540 protein were combined and loaded
onto a 5 ml SP-Sepharose HiTrap column (obtained from Pharmacia
Biotech Inc.) previously equilibrated with 50 mM Tris, 25 mM Sodium
Acetate, 1 mM PMSF, 1 mM .beta.ME, 8 M Urea, pH 4.5 (SP-Sepharose
Buffer). The column was washed with SP-Sepharose Buffer until most
of the unbound protein was removed. Bound protein was eluted with
an increasing salt gradient to 1 M NaCl over 100 ml (20 cv) in
SP-sepharose buffer. Column fractions were analyzed for the
presence of PHIS-PfE6.sub.540 protein by immunoblot using the T7
tag monoclonal antibody as described above. The results indicated
that the PHIS-PfE6.sub.540 protein was eluted at about 0.75 M
NaCl.
[0266] The purified PHIS-PfE6.sub.148 protein was used to produce
an anti-M6 polyclonal antiserum as follows. Rabbits were immunized
with PHIS-PfE6.sub.148 g protein diluted to a concentration of
about 0.1 mg/ml in PBS. One milliliter of the dilution was mixed
1:1 mix with Complete Freunds Adjuvant. In the primary
immunization, about 500 .mu.l of the 1:1 mix was injected
subcutaneously into 5 different sites (0.1 ml/site) and 500 .mu.l
was injected intradermally into 5 different sites (0.1 ml/site) on
the rabbit. Booster shots were administered to the rabbit
intramuscularly in 4 sites using 250 .mu.l/site of a 1:1 mix of
PHIS-PfE6.sub.1488 protein with Incomplete Freunds Adjuvant. The
booster shots were administered at days 14 and 35. Serum samples
were obtained prior to immunization (pre-bleed), and at day 14
after primary immunization and day 14 after the first and second
boost.
[0267] C. Flea esterase protein PHIS-PfE9.sub.528 was produced in
the following maruler. A pair of primers was used to amplify DNA
from flea esterase nucleic acid molecule nfE9.sub.2007 produced as
described in Example 11. A sense primer containing an BamHI site
having the nucleic acid sequence 5'-TTC CGG ATC CGG CTG ATC TAC AAG
TGA CTT TG-3' (denoted SEQ ID NO:64; BamHI site shown in bold), was
used in combination with an anti-sense primer containing a XhoI
site having the nucleic acid sequence 5' TGG TAC TCG AGT CAT AAA
AAT TTA TTC CAA AAT C.sub.3' (denoted SEQ ID NO:65; XhoI site shown
in bold). A PCR product was derived from nfE9.sub.2007, and is
referred to herein as nfE9.sub.1540, having nucleic acid sequence
SEQ ID NO:51. The PCR product was digested with BamI and XhoI
restriction endonucleases, gel purified and subcloned into
expression vector pTrcHisB (available from InVitrogen). The
resultant recombinant molecule, referred to herein as
pTrc-nfE9.sub.540, was transformed into E. coli HB101 competent
cells (available from Gibco BRL) to form recombinant cell E.
coli:pTrc-nfE9.sub.1540.
[0268] The recombinant cells were cultured using the method
described in Section A of this example. Immunoblot analysis of
recombinant cell E. coli:pTrc-nfE9.sub.1540 lysate using a T7 tag
monoclonal antibody directed against the fusion portion of the
recombinant PHIS-PfE9.sub.528 fusion protein identified proteins of
appropriate size, namely an about 59 kD protein for each fusion
protein.
[0269] Expression of the recombinant PHIS-PfE9.sub.528 fusion
protein was improved by transforming supercoiled plasmid
pTrc-nfE9.sub.1584 DNA harvested from E. coli:pTrc-nfE9.sub.1540
cells into the BL-21 strain of E. coli. The amount of expression
PHIS-PfE9.sub.528 was confined by immunoblot using the method
described immediately above.
[0270] Two liters of media from cultures of E. coli cells
expressing PHIS-PfE9.sub.528 protein were harvested and S4
supernatant was prepared using the method described above in
section B. The PHIS-PfE9.sub.528 protein contained in the S4
supernatant was loaded onto a 5.0 ml, Metal chelating HiTrap column
charged with NiCl.sub.2 (available from Pharmacia Biotech Inc.,
Piscataway, N.J.), previously equilibrated with 50 mM Tris, 1 mM
PMSF, 1 mM .beta.ME, 8 M urea, pH 8.0 (Buffer A). The column was
washed with 5 cv of Buffer A until all unbound protein was removed.
Bound protein was eluted with a linear gradient from Buffer A to 50
mM Tris, 1 mM PMSF, 1 mM .beta.ME, 8 M urea, 1 M NaCl, pH 4.0.
Column fractions were analyzed for the presence of
PHIS-PfE9.sub.528 protein by immunoblot using the T7 tag monoclonal
antibody as described above. The results indicated that the
majority of the PHIS-PfE9.sub.528 protein was eluted at about 250
mM NaCl. The fractions containing the PHIS-PfE9.sub.528 protein
were combined and loaded onto a C4-reversed phase column (obtained
from Vydak, Hesperia, Calif.), previously equilibrated with 0.05%
trifluoroacetic acid (TFA). The column was washed with 0.05% TFA
until all unbound protein was removed. Bound proteins were eluted
with a linear gradient from 0.05% TFA to 0.05% TFA in acetonitrile.
Column fractions were analyzed for the presence of
PHIS-PfE9.sub.528 protein by immunoblot using the T7 tag monoclonal
antibody as described above. The results indicated that the
PHIS-PfE9.sub.528 protein was eluted at about 40% acetonitrile. The
fractions containing the PHIS-PfE9.sub.528 protein were combined
and loaded onto a 5 ml Q-Sepharose HiTrap column previously
equilibrated with 50 mM Tris, 25 mM Sodium Acetate, 1 mM PMSF, 1 mM
.beta.ME, 8 M Urea, pH 8.5 (Q-Sepharose Buffer). The column was
washed with Q-Sepharose Buffer until all unbound protein was
removed. Bound protein was eluted with an increasing salt gradient
to 1 M NaCl over 100 ml (20 cv) in Q-sepharose buffer. Column
fractions were analyzed for the presence of PHIS-PfE9.sub.528
protein by immunoblot using the T7 tag monoclonal antibody as
described above. The results indicated that the PHIS-PfE9.sub.528
protein was eluted at about 0.3 M NaCl.
[0271] The purified PHIS-PfE9.sub.528 protein was used to produce
an anti-P 1 polyclonal antiserum as follows. Rabbits were immunized
with PHIS-PfE9.sub.528 protein diluted to a concentration of about
0.1 mg/ml in PBS. One milliliter of the dilution was mixed 1:1 mix
with Complete Freunds Adjuvant. In the primary immunization, about
500 .mu.l of the 1:1 mix was injected subcutaneously into 5
different sites (0.1 ml/site) and 500 .mu.l was injected
intradermally into 5 different sites (0.1 ml/site) on the rabbit.
Booster shots were administered to the rabbit intramuscularly in 4
sites using 250 .mu.l/site of a 1:1 mix of PHIS-PfE9.sub.528
protein with Incomplete Freunds Adjuvant The booster shots were
administered at days 14 and 35. Serum samples were obtained prior
to immunization (pre-bleed), and at day 14 after primary
immunization and day 14 after the first and second boost.
[0272] D. Flea esterase protein PHIS-PfE7.sub.275 was produced in
the following manner. A 650-bp fragment was produced by digesting
nfE7.sub.2836 DNA with the restriction enzymes BamHI and BglII. The
BamHI and BglII fragment derived from nfE7.sub.2836 is referred to
herein as nfE7.sub.650, having nucleic acid sequence SEQ ID NO:72
and amino acid SEQ ID NO:73. The fragment was purified using a
Qiaquick.TM. Kit (available from Qiagen, Santa Clarita, Calif.),
according to methods provided by the manufacturer. The purified
fragment was subcloned into expression vector pTrcHisC which had
been digested with BamHI and BglII. The resultant recombinant
molecule, referred to herein as pTrc-nfE7.sub.650 was transformed
into E. coli DH-5a competent cells (available from Gibco BRL) to
form recombinant cell E. coli:pTrc-nfE7.sub.650.
[0273] The recombinant cells were cultured using the method
described above in section A. Immunoblot analysis of recombinant
cell E. coli:pTrc-nfE7.sub.650 lysate using a T7 tag monoclonal
antibody directed against the fusion portion of the recombinant
PHIS-PfE7.sub.275 fusion protein identified proteins of appropriate
size, namely an about 35 kD protein for each fusion protein.
[0274] Expression of the recombinant fusion protein was improved by
transforming supercoiled plasmid pTrc-nfE7.sub.650 DNA harvested
from E. coli:pTrc-nfE7.sub.650 cells into the BL-21 strain of E.
coli. The amount of expression E. coli:pTrc-nfE7.sub.650 was
confined by immunoblot using the method described immediately
above.
Example 13
[0275] This Example demonstrates the production of esterase
proteins of the present invention in eukaryotic cells.
[0276] A. Recombinant molecule pBv-nfE7.sub.1788, containing a flea
esterase nucleic acid molecule spanning nucleotides from about 99
through about 1886 of SEQ ID NO:24, and pBv-nfE8.sub.1785,
containing a flea esterase nucleic acid molecule spanning
nucleotides from about 99 through about 1883 of SEQ ID NO:30 each,
operatively linked to baculovirus polyhedron transcription control
sequences were produced in the following manner. In order to
subclone a flea esterase nucleic acid molecule into baculovirus
expression vectors, flea esterase nucleic acid molecule-containing
fragments were separately PCR amplified from nfE7.sub.2836 or
nfE8.sub.2801 DNA. A PCR fragment of 1858 nucleotides, named
nfE7.sub.1858, was amplified from nfE7.sub.2836 using a sense
primer E1113 FWD having the nucleic acid sequence 5'-AAAACTGCAG
TATAAATATG TTACCTCACA GTAGTG-3' (SEQ ID NO:49; PstI site shown in
bold) and an antisense primer E 1113/2212 REV having the nucleic
acid sequence 5'-TGCTCTAGAT TATCTAATAC TTCCTTCATT ACAG (SEQ ID
NO:50; XbaI site shown in bold). A PCR fragment of 1858
nucleotides, named nfE8.sub.1858, was amplified from nfE8.sub.2801
using a sense primer E2212 FWD having the nucleic acid sequence
5'-AAAACTGCAG TATAAATATG TTACCTCACA GTGCATTAG-3' (SEQ ID NO:66;
PstI site shown in bold), and the antisense primer E 1113/2212 REV.
The N-terminal primer was designed from the pol h sequence of
baculovirus with modifications to enhance expression in the
baculovirus system.
[0277] In order to produce a baculovirus recombinant molecule
capable of directing the production of PfE7.sub.596, the about
1,802 base pair PCR product (referred to as Bv-nfE7.sub.1802) was
digested with PstI and XbaI and subcloned into unique PstI and XbaI
sites of pVL1392 baculovirus shuttle plasmid (available from
Pharmingen, San Diego, Calif.) to produce the recombinant molecule
referred to herein as pVL-nfE7.sub.1802.
[0278] In order to produce a baculovirus recombinant molecule
capable of directing the production of PfE8.sub.595, the about
1,792 base pair PCR product (referred to as Bv-nfE8.sub.1792) was
digested with PstI and XbaI and subcloned into PstI and XbaI
digested to produce the recombinant molecule referred to herein as
pVL-nfE8.sub.1792.
[0279] The resultant recombinant molecules, pVL-nfE7.sub.1902 and
pVL-nfE8.sub.1792, were verified for proper insert orientation by
restriction mapping. Such a recombinant molecule can be
co-transfected with a linear Baculogold baculovirus DNA (available
from Pharmingen) into S. frugiperda Sf9 cells (available from
InVitrogen) to form the recombinant cells denoted S.
frugiperda:pVL-nfE7.sub.1802 and S. frugiperda:pVL-nfE8.sub.1792.
S. frugiperda:pVL-nfE7.sub.1802 can be cultured in order to produce
a flea esterase protein PfE7.sub.596. S.
frugiperda:pVL-nfE8.sub.1792 can be cultured in order to produce a
flea esterase protein PfE8.sub.595.
[0280] B. Recombinant molecule pBv-PfE9.sub.528, containing a flea
esterase nucleic acid molecule spanning nucleotides from 14 through
1595 of SEQ ID NO:36, operatively linked to baculovirus polyhedron
transcription control sequences were produced in the following
manner. In order to subclone a flea esterase nucleic acid molecule
into baculovirus expression vectors, a flea esterase nucleic acid
molecule-containing fragment was PCR amplified from nfE9.sub.2007
DNA. A PCR fragment of about 1600 nucleotides, named nfE9.sub.1600,
was amplified from nfE9.sub.2007 using a sense primer P121 B1 Sense
having the nucleic acid sequence 5'-CGC GGA TCC GCT GAT CTA CAA GTG
ACT TTG C-3' (SEQ ID NO:75; BamHI site shown in bold) and an
antisense primer P121B1 Anti having the nucleic acid sequence
5'-CCG AGC GGC CGC ATA AAA ATT TAT TCC AAA ATC TAA GTC G-3' (SEQ ID
NO:76; NotI site shown in bold). The N-terminal primer was designed
from the pol h sequence of baculovirus with modifications to
enhance expression in the baculovirus system.
[0281] In order to produce a baculovirus recombinant molecule
capable of directing the production of PfE9.sub.528, the about
1,600 base pair PCR product (referred to as Bv-nfE9.sub.1600) w %
as digested with BamHI and NotI and subcloned into unique BamHI and
NotI sites of pVL1393 baculovirus shuttle plasmid (available from
Pharmingen, San Diego, Calif.) to produce the recombinant molecule
referred to herein as pVL-nfE9.sub.1600.
[0282] The resultant recombinant molecule, pVL-nfE9.sub.1600, was
verified for proper insert orientation by restriction mapping. Such
a recombinant molecule can be co-transfected with a linear
Baculogold baculovirus DNA into S. frugiperda Sf9 cells to form the
recombinant cells denoted S. frugiperda:pVL-nfE9.sub.1600. S.
frugiperda:pVL-nfE9.sub.1600 can be cultured in order to produce a
flea esterase protein PfE9.sub.528.
[0283] An immunoblot of supernatant from cultures of S.
frugiperda:pVL-nfE9.sub.1600 cells producing the flea esterase
protein PfE9.sub.528 was performed using the anti-P1 polyclonal
antiserum described in detail in Example 12. Blots were incubated
using serum samples from the pre-bleed or from serum collected 14
days after the first boost of the rabbit. Analysis of the
supernatent from cultures of S. frugiperda:pVL-nfE9.sub.1600 cells
identified an about 66 kD protein
[0284] C. Recombinant molecule pBv-PfE6.sub.530, containing a flea
esterase nucleic acid molecule spanning nucleotides from 50 through
1701 of SEQ ID NO:18, operatively linked to baculovirus polyhedron
transcription control sequences were produced in the following
manner. In order to subclone a flea esterase nucleic acid molecule
into baculovirus expression vectors, a flea esterase nucleic acid
molecule-containing fragment was PCR amplified from nfE6.sub.1792
DNA. A PCR fragment of about 1679 nucleotides, named
nfE10.sub.1679, was amplified from nfE6.sub.1792 using a sense
primer M6M32 Sense having the nucleic acid sequence 5'-GCG AGG CCT
TAT AAA TAT GTC TCG TGT TAT TTT TTT AAG TTG-3' (SEQ ID NO:75; StuI
site shown in bold) and an antisense primer M6M32 Anti having the
nucleic acid sequence 5'-GCA CTG CAG TTA TTG ACT GTG CAA AGT TTT
TGT GG-3' (SEQ ID NO:76; PstI site shown in bold). The N-terminal
primer was designed from the pol h sequence of baculovirus with
modifications to enhance expression in the baculovirus system.
[0285] In order to produce a baculovirus recombinant molecule
capable of directing the production of PfE6.sub.530, the about
1,679 base pair PCR product (referred to as Bv-nfE6.sub.1679) was
digested with StuI and PstI and subcloned into unique StuI and PstI
sites of FAST BAC.TM. baculovirus shuttle plasmid (obtained from
Gibco-BRL) to produce the recombinant molecule referred to herein
as pFB-nfE6.sub.1679.
[0286] The resultant recombinant molecule, pFB-nfE6.sub.1679, was
verified for proper insert orientation by restriction mapping. Such
a recombinant molecule can be transformed into E. coli strain DH10
(obtained from Gibco-BRL) according to the manufacturer's
instructions. The pFB-nfE6.sub.1679 isolated from the transformed
DH10 cells can then be co-transfected with a linear Baculogold
baculovirus DNA into S. frugiperda Sf9 cells to form the
recombinant cells denoted S. frugiperda:pFB-nfE6.sub.1679. S.
frugiperda:pFB-nfE6,679 can be cultured in order to produce a flea
esterase protein PfE6.sub.530.
[0287] An immunoblot of supernatant from cultures of S.
frugiperda:pFB-nfE6.sub.1679 cells producing the flea esterase
protein PfE6.sub.530 was performed using the anti-M6 polyclonal
antiserum described in detail in Example 12. Blots were incubated
using serum samples from the pre-bleed or from serum collected 14
days after the first boost of the rabbit. Analysis of the
supernatent from cultures of S. frugiperda:pFB-nfE6.sub.1679 cells
identified an about 66 kD protein.
[0288] N-terminal amino acid sequence was obtained using standard
methods for the about 66 kD protein identified using the anti-M6
polyclonal antiserum. The N-terminal amino acid sequence was
determined to be identical to the N-terminal amino acid sequence of
SEQ ID NO:44.
Example 14
[0289] This example describes the purification of carboxylesterase
protein from fed flea midguts.
[0290] About 43,000 cat blood-fed adult flea midguts were collected
and prepared as previously described in Example 1. The extract was
then added in 2 aliquots to columns containing about 1 to about 2
ml of p-aminobenzamidine linked agarose beads (available from
Sigma), equilibrated in 50 mM Tris (pH 8.0), 400 mM NaCl, and
incubated overnight at 4.degree. C. The columns were then drained
to remove unbound protein and the two aliquots of unbound protein
were combined. The collected unbound protein was then concentrated
and diafiltered into a total volume of about 16 ml of 25 mM Tris
(pH 8), 10 mM NaCl using an Ultrafree-20 10 kD centrifugal
concentrator (available from Millipore, Bedford, Mass.).
[0291] Aliquots of about 8 ml were loaded onto an Uno Q6 anion
exchange column (available from Bio-Rad, Hercules, Calif.)
equilibrated in 25 mM Tris (pH 8), 10 mM NaCl, operated on a
BioLogic liquid chromatography system (available from Bio-Rad). The
column was washed with 25 mM Tris (pH 8), 10 mM NaCl until all
unbound protein was removed. Protein bound to the column was then
eluted with a linear gradient from 10 mM to 1 M NaCl in 25 mM Tris,
pH 8. Fractions were assayed for CE activity using the assay
described previously. The results indicated that CE activity was
eluted at about 220 mM NaCl.
[0292] Fractions containing CE activity were pooled and diafiltered
into a total volume of about 3 ml of 20 mM MES buffer
(2-(N-morpholino)ethanesul- fonic acid), pH 6.0, containing 10 mM
NaCl, in preparation for cation exchange chromatography. The sample
was then applied to an Uno SI cation exchange column (available
from Bio-Rad) equilibrated in MFS buffer. The column was washed
with MES buffer until all unbound protein was removed. Protein
bound to the column was then eluted with a linear gradient from 10
mM to 1 M NaCl in 20 mM MES buffer, pH 6. Fractions were assayed
for CE activity using the assay described previously. The results
indicated that CE activity was not retained on the cation exchange
column using the above conditions, and all of the activity was
found in the flow-through fractions.
[0293] Fractions containing CE activity were pooled and diafiltered
into a total volume of about 3 ml of 25 mM Tris (pH 8), 10 mM NaCl,
in preparation for an additional anion exchange chromatography
step. The sample was then applied to a Bio-Scale Q2 anion exchange
column (available from Bio-Rad). The column was washed with 25 mM
Tris (pH 8), 10 mM NaCl until all unbound protein was removed.
Protein bound to the column was then eluted with a linear gradient
from 10 mM to 1 M NaCl in 25 mM Tris, pH 8. Fractions were assayed
for CE activity using the assay described previously. The results
indicated that CE activity was eluted at about 130 mM NaCl.
[0294] A fraction containing CE activity was diluted into a total
volume of about 4 ml of 10 mM phosphate buffer, pH 7.2 containing
10 mM NaCl, in preparation for hydroxyapatite chromatography. The
sample was then applied to a Bio-Scale CHT2-I column (available
from Bio-Rad) at a flow rate of about 0.5 ml/min. The column was
washed with 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCl
until all unbound protein was removed. Protein bound to the column
was then eluted with a linear gradient from 10 mM phosphate buffer,
pH 7.2 containing 10 mM NaCl to 0.5 M 10 nM phosphate buffer, pH
6.5 containing 10 mM NaCl. Fractions were assayed for CE activity
using the assay described previously. The results indicated that CE
activity was eluted at about 200 mM phosphate.
Example 15
[0295] This example describes the purification of a
carboxylesterase protein from wandering flea larvae.
[0296] About 120,000 bovine blood-fed adult wandering flea larvae
were homogenized in 3 batches of about 40,000 wandering larvae in
each batch, in Tris buffered saline (TBS), pH 8.0 as previously
described, except that about 1.2 mg of phenylthiourea was added to
each ml of TBS during the extraction procedure to inhibit cross
linking reactions. The extracts were dialyzed against 2 changes of
about 2 L of 10 mM phosphate buffer, pH 7.2 containing 10 mM NaCl
in preparation for hydroxyapatite batch chromatography. The samples
were then filtered through glass Acrodiscs.RTM. (available from
Gelman Sciences, Ann Arbor, Mich.) and added to 14 g of Macro-Prep
Ceramic Hydroxyapatite, Type 1,40 .mu.m beads (available from
Bio-Rad), previously equilibrated in 10 mM phosphate buffer, pH 7.2
containing 10 mM NaCl. The extracts and beads were rocked at room
temperature for about 30 minutes. Following incubation, the beads
were centrifuged for about 5 minutes at 500.times.g and the
supernatants removed. The beads were washed with about 40 ml 10 mM
phosphate buffer, pH 7.2 containing 10 mM NaCl, centrifuged as
above, and washed and centrifuged again to eliminate all unbound
protein. Bound proteins were eluted by washing the beads with about
40 ml of each of-100 mM, 200 mM, 300 mM, and 400 mM phosphate
buffer, pH 6.5 containing 10 mM NaCl. Following elution, the
supernatants from each concentration of phosphate buffer were
tested for juvenile hormone esterase activity as described
previously in Example 7. The juvenile hormone esterase activity
eluted at different phosphate concentrations in each batch, but the
activity was generally found in the 200 mM to 300 mM phosphate
fractions.
[0297] The fractions that contained the highest juvenile hormone
esterase activity were combined and diafiltered into a total volume
of about 50 ml of 10 mM phosphate buffer, pH 7.2 containing 10 mM
NaCl using a stirred cell concentrator fitted with a YM10
ultrafiltration membrane (available from Amicon, Beverly, Mass.).
Aliquots of about 5 ml to 10 ml were applied to a chromatography
column containing about 10 ml of Macro-Prep Ceramic Hydroxyapatite,
Type 1,20 .mu.m beads, previously equilibrated with 10 mM phosphate
buffer, pH 7.2 containing 10 mM NaCl. The column was washed with 10
mM phosphate buffer, pH 7.2 containing 10 mM NaCl until all unbound
protein was removed. Protein bound to the column was then eluted
with a linear gradient from 10 mM phosphate buffer, pH 7.2
containing 10 mM NaCl to 0.5 M 10 mM phosphate buffer, pH 6.5
containing 10 mM NaCl. Fractions were assayed for carboxylesterase
activity using the assay described previously. The results
indicated that carboxylesterase activity was eluted at about 160 mM
phosphate.
[0298] The fractions that contained the highest carboxylesterase
activity w ere combined and diafiltered into a total volume of
about 15 ml of 20 mM sodium acetate buffer, pH 4.0 in preparation
for cation exchange chromatography. Aliquots of about 3 ml were
applied to a PolyCat A cation exchange column (available from
PolyLC, Columbia, Md.) equilibrated in 20 mM sodium acetate buffer,
pH 6.0, operated on a Waters high performance liquid chromatography
system (available from Waters Corporation, Milford, Mass.). The
column was washed with 20 mM sodium acetate buffer, pH 6.0 until
all unbound protein was removed. Protein bound to the column was
then eluted with a linear gradient from 20 mM sodium acetate
buffer, pH 6.0 to 20 mM sodium acetate buffer, pH 6.0 containing 1
M NaCl. Fractions were assayed for CE activity using the assay
described previously. The results indicated that there were two
pools of CE activity. The first pool was not retained on the cation
exchange column, and the second pool was eluted at about 170 mM
NaCl.
[0299] The fractions from the second pool that contained the
highest carboxylesterase activity were combined and diafiltered
into a total volume of about 10 ml of 25 mM Tris (pH 8), 10 mM
NaCl, in preparation for anion exchange chromatography. The sample
was then applied to a Bio-Scale Q2 anion exchange column (available
from Bio-Rad). The column was washed with 25 mM Tris (pH 8), 10 mM
NaCl until all unbound protein was removed. Protein bound to the
column was then eluted with a linear gradient from 10 mM to 1 M
NaCl in 25 mM Tris, pH S. Fractions were assayed for
carboxylesterase activity using the assay described previously. The
results indicated that carboxylesterase activity was eluted at
about 350 mM NaCl.
[0300] Fractions containing carboxylesterase activity were combined
and concentrated to about 175 .mu.l using a Centricon 10
centrifugal concentrator (available from Amicon, Beverly, Mass.) in
preparation for size exclusion chromatography. The sample was
applied to a Bio-Select SEC 125-5 size exclusion chromatography
column (available from Bio-Rad), previously equilibrated in TBS, pH
7.2. About 250 .mu.l fractions were then collected. The fractions
were assayed for carboxylesterase activity using the assay
described previously. The results indicated that the
carboxylesterase activity was eluted in about 5.5 to uiil of
buffer, corresponding to a molecular weight of about 40 to 100 kDa
based on the elution volumes of gel filtration molecular weight
standard proteins (available from Sigma, St. Louis, Mo.).
Example 16
[0301] This example describes the purification of juvenile hormone
esterase activity from unfed adult flea midguts by affinity
chromatography.
[0302] About 16,000 unfed adult flea midguts were collected in 20
mM Tris buffer (pH 7.7), containing 130 mM NaCl, 1 mM sodium EDTA,
1 mM Pefabloc.RTM. (available from Boehringer Mannheim,
Indianapolis, Ind.), 1 microgram/ml (.mu.g/ml) leupeptin and 1
.mu.g/ml pepstatin. The midguts were homogenized by freeze-fracture
and sonication, and then centrifuged at about 14,000.times. g for
20 min. The soluble material from the centrifugation step was
recovered, diafiltered into Tris buffered saline (TBS), and applied
to a disposable plastic column containing about 1 ml of
3-[(4'-mercapto)butylthio]-1,1,1-trifluoropropan-2-one linked
Sepharose 6B beads, prepared similarly to the method described by
Venkateshi et al. (J. Biol. Chem., Vol. 265, No. 35, 21727-21732,
1990) (the 3-[(4'-mercapto)butylthio]-1,1,1-trifluoropropan-2-one
was a gift from Novartis Corp., Basel, Switzerland; and the
Epoxy-activated Sepharose 6B is available from Pharmacia Biotech
Inc., Piscataway, N.J.). After overnight incubation at 4.degree.
C., the column was drained and the beads were washed with about 10
ml TBS, then about 10 ml TBS containing 0.1% (w/v) n-octylglucoside
(OG; available from Boehringer Mannheim). The pre-column,
flow-through, and wash fractions were tested for juvenile hormone
esterase activity by the method previously described above in
Example 7. The results indicate that the flow-through fraction
contained approximately 40% less juvenile hormone esterase activity
than the pre-column material, and that the washes contained very
little activity.
[0303] Bound protein was eluted from the beads by adding about 10
ml of TBS containing 0.1% (w/v) OG and 1 mM
3-octylthio-1,1,1-trifluoropropan-2- -one (OTFP; a gift from
Novartis Corp.). After a 2 hour incubation at 4.degree. C., about 5
ml of the eluate was collected, and the remaining 5 ml was
incubated with the beads overnight at 4.degree. C. The following
day, the beads were drained, the eluate collected, and an
additional 10 ml of TBS containing 0.1% (w/v) OG and 1 mM OTFP was
added to the beads. After an overnight incubation at 4.degree. C.,
the beads were drained and the eluate collected. The final 10 ml
elution step was repeated 3 additional times so that we had 6
eluted fractions. The first elution fraction was dialyzed overnight
twice against 1 liter of fresh TBS to remove excess OTFP. The
second elution fraction was also dialyzed overnight against 1 liter
of fresh TBS to remove OTFP. The third through sixth elution
fractions were not dialyzed. All six eluted fractions were tested
for juvenile hormone esterase activity by the method previously
described above in Example 7. The results indicate that only the
third elution fraction contained detectable juvenile hormone
esterase activity. Analysis of the eluted fractions by
silver-stained SDS-PAGE indicated that several proteins were
specifically bound to the affinity beads and were eluted by OTFP.
The apparent molecular weights of these proteins, as determined by
SDS-PAGE, were about 66 kDa, 55 kDa, and 33 kDa. About 3.5 ml of
each elution fraction were combined and concentrated to about 110
.mu.l using a Centriplus 10 centrifugal concentrator (available
from Anicon, Beverly, Mass.). This pool was separated by SDS-PAGE
and blotted onto a polyvinylidene difluoride (PVDF) membrane as
described previously in Example 5. The stained protein band at
about 66 kDa was excised and subjected to N-terminal sequence
analysis as described previously.
[0304] The results indicated that the N-terminal amino acid
sequence of the putative 66 kDa juvenile hormone esterase protein
was DL y/g V k/y/g v/q/n LQGTLKGKE (denoted herein as SEQ ID
NO:74), in which the lower case letters designate uncertainties.
Below is shown a comparison between different esterase amino acid
sequences of the present invention.
1 SEQ ID NO:74: DL (y/g) V (k/y/g) (v/q/n) LQGTLKGKE SEQ ID NO:37:
DL Q V T L LQGTLKGKE (Residues 3-17)
Example 17
[0305] This example describes the purification of an active
recombinant juvenile hormone esterase protein from baculovirus
supernatants.
[0306] About 1 liter of supernatant from cultures of S.
frugiperda:pVL-nfE9.sub.1600 cells producing the flea esterase
protein PfE9.sub.528 was brought to about 50% salutation with
ammonium sulfate and centrifuged at about 20000.times.g for about
30 minutes at 4.degree. C. to pellet the precipitated material.
After centrifugation, the pellet was retained and the supernatant
was brought to about 100% saturation with ammonium sulfate and
centrifuged as above. The material in both pellets were resuspended
separately in about 35 ml of Tris buffered saline (TBS), pH 8.0.
The resuspended pellets were assayed for the presence of flea
esterase protein PfE9.sub.528 g using standard Western blot
techniques and a polyclonal antiserum that binds specifically to
PfE9.sub.528 protein. Briefly, a rabbit was immunized with
PHIS-PfE9.sub.528 protein purified from E. coli:pTrc-nfE9.sub.1584
cells (described above in Example 12C) and boosted using standard
procedures. The results indicated that the flea esterase protein
PfE9.sub.528 was present in the S. frugiperda:pVL-nfE9.sub.1600
supernatants and the protein was precipitated by adjusting the
ammonium sulfate concentration from about 50% saturation to about
100% saturation.
[0307] The resuspended flea protein PfE9.sub.528 was diafiltered
into about 10 ml of 25 mM Tris (pH 8.0), 10 mM NaCl using an
Ultrafree-20 10 kD centrifugal concentrator in preparation for
anion exchange chromatography. Aliquots of about 5 ml were loaded
onto an Uno Q6 anion exchange column equilibrated in 25 mM Tris (pH
8.0), 10 mM NaCl. The column was washed with 25 mM Tris (pH 8.0),
10 mM NaCl until most of the unbound protein was removed. Protein
bound to the column was then eluted with a linear gradient from 10
mM to 1 M NaCl in 25 mM Tris buffer (pH 8.0). Fractions were
assayed for the presence of flea esterase protein PfE9.sub.528 by
the immunoblot method described above. The results indicated that
the flea esterase protein PfE9.sub.528 was eluted at about 200 mM
NaCl.
[0308] Fractions containing the flea esterase protein PfE9.sub.528
were pooled and concentrated to about 440 .mu.l using a Centricon
10 kD centrifugal concentrator in preparation for size exclusion
chromatography. The sample was applied in 3 aliquots to a
Bio-Select SEC 125-5 size exclusion chromatography column
(available from Bio-Rad), previously equilibrated in TBS, pH 7.2.
The column was eluted with TBS, pH 7.2 at a flow rate of about 0.5
ml/min, and fractions of about 250 .mu.l were collected. The
fractions were assayed for the presence of flea esterase protein
PfE9.sub.528 by the immunoblot method described above. The results
indicated that the flea esterase protein PfE9.sub.528 was eluted
with about 6 ml of buffer, corresponding to a molecular weight of
about 40 to 100 kDa based on the elution volumes of gel filtration
molecular weight standard proteins (available from Sigma, St.
Louis, Mo.).
[0309] Fractions containing flea esterase protein PfE9.sub.528 were
then assayed for juvenile hormone esterase activity as described in
Example 7 and carboxylesterase activity as described in Example 2.
The results indicated that the purified flea esterase protein
PfE9.sub.528 had both juvenile hormone esterase activity and
carboxylesterase activity.
[0310] 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 1
1
76 1 401 DNA Ctenocephalides felis CDS (92)..(400) 1 tttacatcat
taataaacat aaatctaata aatcttgtgg atcaagatca agtttattag 60
tgagagtgtt ggatttgtga aatatttcaa a atg aat tct tta att gta aaa 112
Met Asn Ser Leu Ile Val Lys 1 5 att tct caa gga gct att gag ggg aag
gaa atg att aat gat aat gga 160 Ile Ser Gln Gly Ala Ile Glu Gly Lys
Glu Met Ile Asn Asp Asn Gly 10 15 20 aag tcg ttt aga gga ttt ttg
ggt ata cct tat gct aaa ccg cct ata 208 Lys Ser Phe Arg Gly Phe Leu
Gly Ile Pro Tyr Ala Lys Pro Pro Ile 25 30 35 gga aat ctt ana ttt
aag cct cct caa aag cct gat gat tgg aat gat 256 Gly Asn Leu Xaa Phe
Lys Pro Pro Gln Lys Pro Asp Asp Trp Asn Asp 40 45 50 55 gtt cga cca
gct act gaa naa gca aat ggt tgt aga tcg aaa cat atg 304 Val Arg Pro
Ala Thr Glu Xaa Ala Asn Gly Cys Arg Ser Lys His Met 60 65 70 ctg
cag cat cat att att gga gac naa nat tgt cta tac cta aac gtn 352 Leu
Gln His His Ile Ile Gly Asp Xaa Xaa Cys Leu Tyr Leu Asn Val 75 80
85 tat gtt cca ttg act tcc aaa ttg gag aaa cta cca gta atg ttc tgg
g 401 Tyr Val Pro Leu Thr Ser Lys Leu Glu Lys Leu Pro Val Met Phe
Trp 90 95 100 2 103 PRT Ctenocephalides felis misc_feature
(43)..(43) The 'Xaa' at location 43 stands for Lys, Arg, Thr, or
Ile. 2 Met Asn Ser Leu Ile Val Lys Ile Ser Gln Gly Ala Ile Glu Gly
Lys 1 5 10 15 Glu Met Ile Asn Asp Asn Gly Lys Ser Phe Arg Gly Phe
Leu Gly Ile 20 25 30 Pro Tyr Ala Lys Pro Pro Ile Gly Asn Leu Xaa
Phe Lys Pro Pro Gln 35 40 45 Lys Pro Asp Asp Trp Asn Asp Val Arg
Pro Ala Thr Glu Xaa Ala Asn 50 55 60 Gly Cys Arg Ser Lys His Met
Leu Gln His His Ile Ile Gly Asp Xaa 65 70 75 80 Xaa Cys Leu Tyr Leu
Asn Val Tyr Val Pro Leu Thr Ser Lys Leu Glu 85 90 95 Lys Leu Pro
Val Met Phe Trp 100 3 401 DNA Ctenocephalides felis misc_feature
(50)..(50) n = unknown 3 cccagaacat tactggtagt ttctccaatt
tggaagtcaa tggaacatan acgtttaggt 60 atagacaatn ttngtctcca
ataatatgat gctgcagcat atgtttcgat ctacaaccat 120 ttgcttnttc
agtagctggt cgaacatcat tccaatcatc aggcttttga ggaggcttaa 180
atntaagatt tcctataggc ggtttagcat aaggtatacc caaaaatcct ctaaacgact
240 ttccattatc attaatcatt tccttcccct caatagctcc ttgagaaatt
tttacaatta 300 aagaattcat tttgaaatat ttcacaaatc caacactctc
actaataaac ttgatcttga 360 tccacaagat ttattagatt tatgtttatt
aatgatgtaa a 401 4 364 DNA Ctenocephalides felis CDS (2)..(364) 4 g
tct cgt gtt att ttt tta agt tgt att ttt ttg ttt agt ttt aat ttt 49
Ser Arg Val Ile Phe Leu Ser Cys Ile Phe Leu Phe Ser Phe Asn Phe 1 5
10 15 ata aac tgt gat tcc ccg act gta act ttg ccc caa ggc gaa ttg
gtt 97 Ile Asn Cys Asp Ser Pro Thr Val Thr Leu Pro Gln Gly Glu Leu
Val 20 25 30 gga aaa gct ttg acg aac gaa aat gga aaa gag tat ttt
agc tac aca 145 Gly Lys Ala Leu Thr Asn Glu Asn Gly Lys Glu Tyr Phe
Ser Tyr Thr 35 40 45 ggt gta cct tat gct aaa cct cct gtt gga gaa
ctt aga ttt aag cct 193 Gly Val Pro Tyr Ala Lys Pro Pro Val Gly Glu
Leu Arg Phe Lys Pro 50 55 60 cca cag aaa gct gag cca tgg caa ggt
gtt ttc aac gcc aca tta tac 241 Pro Gln Lys Ala Glu Pro Trp Gln Gly
Val Phe Asn Ala Thr Leu Tyr 65 70 75 80 gga aat gtg tgt aaa tct tta
aat ttc ttc ttg aag aaa att gaa gga 289 Gly Asn Val Cys Lys Ser Leu
Asn Phe Phe Leu Lys Lys Ile Glu Gly 85 90 95 gac gaa gac tgc ttg
gta gta aac gtg tac gca cca aaa aca act tct 337 Asp Glu Asp Cys Leu
Val Val Asn Val Tyr Ala Pro Lys Thr Thr Ser 100 105 110 gat aaa aaa
ctt cca gta ttt ttc tgg 364 Asp Lys Lys Leu Pro Val Phe Phe Trp 115
120 5 121 PRT Ctenocephalides felis 5 Ser Arg Val Ile Phe Leu Ser
Cys Ile Phe Leu Phe Ser Phe Asn Phe 1 5 10 15 Ile Asn Cys Asp Ser
Pro Thr Val Thr Leu Pro Gln Gly Glu Leu Val 20 25 30 Gly Lys Ala
Leu Thr Asn Glu Asn Gly Lys Glu Tyr Phe Ser Tyr Thr 35 40 45 Gly
Val Pro Tyr Ala Lys Pro Pro Val Gly Glu Leu Arg Phe Lys Pro 50 55
60 Pro Gln Lys Ala Glu Pro Trp Gln Gly Val Phe Asn Ala Thr Leu Tyr
65 70 75 80 Gly Asn Val Cys Lys Ser Leu Asn Phe Phe Leu Lys Lys Ile
Glu Gly 85 90 95 Asp Glu Asp Cys Leu Val Val Asn Val Tyr Ala Pro
Lys Thr Thr Ser 100 105 110 Asp Lys Lys Leu Pro Val Phe Phe Trp 115
120 6 364 DNA Ctenocephalides felis 6 ccagaaaaat actggaagtt
ttttatcaga agttgttttt ggtgcgtaca cgtttactac 60 caagcagtct
tcgtctcctt caattttctt caagaagaaa tttaaagatt tacacacatt 120
tccgtataat gtggcgttga aaacaccttg ccatggctca gctttctgtg gaggcttaaa
180 tctaagttct ccaacaggag gtttagcata aggtacacct gtgtagctaa
aatactcttt 240 tccattttcg ttcgtcaaag cttttccaac caattcgcct
tggggcaaag ttacagtcgg 300 ggaatcacag tttataaaat taaaactaaa
caaaaaaata caacttaaaa aaataacacg 360 agac 364 7 421 DNA
Ctenocephalides felis CDS (113)..(421) 7 tttacattac atcaaatcat
atttttatta gtatattttt tagaagaacc tagccaaaaa 60 atatggactt
tagactgtga ttaatttatt ttacctgaga ttttccttta ca atg ggt 118 Met Gly
1 gat ctt caa gtg act ttg tta caa ggt tct ttg aga gga aaa gag caa
166 Asp Leu Gln Val Thr Leu Leu Gln Gly Ser Leu Arg Gly Lys Glu Gln
5 10 15 att aat gaa aag gga aat gtg ttt tat agt tat tct gga att cca
tat 214 Ile Asn Glu Lys Gly Asn Val Phe Tyr Ser Tyr Ser Gly Ile Pro
Tyr 20 25 30 gcc aaa cct cca gtt ggt gat cta aga ttc aag cca cct
caa cct gca 262 Ala Lys Pro Pro Val Gly Asp Leu Arg Phe Lys Pro Pro
Gln Pro Ala 35 40 45 50 gaa cct tgg tca ggt gtc ctt gat gct act aaa
gaa ggg aat agt tgt 310 Glu Pro Trp Ser Gly Val Leu Asp Ala Thr Lys
Glu Gly Asn Ser Cys 55 60 65 aga tct gta cat ttt att aaa aag att
aaa gta ggg gct gaa gat tgt 358 Arg Ser Val His Phe Ile Lys Lys Ile
Lys Val Gly Ala Glu Asp Cys 70 75 80 cta tac ctc aat gtc tat gta
cca aaa aca tca gag aaa tcc ctt ctt 406 Leu Tyr Leu Asn Val Tyr Val
Pro Lys Thr Ser Glu Lys Ser Leu Leu 85 90 95 cca gta atg gta tgg
421 Pro Val Met Val Trp 100 8 103 PRT Ctenocephalides felis 8 Met
Gly Asp Leu Gln Val Thr Leu Leu Gln Gly Ser Leu Arg Gly Lys 1 5 10
15 Glu Gln Ile Asn Glu Lys Gly Asn Val Phe Tyr Ser Tyr Ser Gly Ile
20 25 30 Pro Tyr Ala Lys Pro Pro Val Gly Asp Leu Arg Phe Lys Pro
Pro Gln 35 40 45 Pro Ala Glu Pro Trp Ser Gly Val Leu Asp Ala Thr
Lys Glu Gly Asn 50 55 60 Ser Cys Arg Ser Val His Phe Ile Lys Lys
Ile Lys Val Gly Ala Glu 65 70 75 80 Asp Cys Leu Tyr Leu Asn Val Tyr
Val Pro Lys Thr Ser Glu Lys Ser 85 90 95 Leu Leu Pro Val Met Val
Trp 100 9 421 DNA Ctenocephalides felis 9 ccataccatt actggaagaa
gggatttctc tgatgttttt ggtacataga cattgaggta 60 tagacaatct
tcagccccta ctttaatctt tttaataaaa tgtacagatc tacaactatt 120
cccttcttta gtagcatcaa ggacacctga ccaaggttct gcaggttgag gtggcttgaa
180 tcttagatca ccaactggag gtttggcata tggaattcca gaataactat
aaaacacatt 240 tcccttttca ttaatttgct cttttcctct caaagaacct
tgtaacaaag tcacttgaag 300 atcacccatt gtaaaggaaa atctcaggta
aaataaatta atcacagtct aaagtccata 360 ttttttggct aggttcttct
aaaaaatata ctaataaaaa tatgatttga tgtaatgtaa 420 a 421 10 524 DNA
Ctenocephalides felis CDS (113)..(523) 10 gaacgttgat acgatagaca
tgtcgtcttc aaaacgtcta ttttatcata aacaaaacga 60 gataaataat
aacaattaag caaccaaaat gcattaaaaa acacaataaa aa atg tta 118 Met Leu
1 cct cac agt agt gca tta gtt tta ttt tta ttt ttt tta ttt ttc tta
166 Pro His Ser Ser Ala Leu Val Leu Phe Leu Phe Phe Leu Phe Phe Leu
5 10 15 ttt aca cct atc ttg tgc ata cta tgg gat aac cta gat cag cat
ttg 214 Phe Thr Pro Ile Leu Cys Ile Leu Trp Asp Asn Leu Asp Gln His
Leu 20 25 30 tgc aga gtt caa ttt aac agg atc acg gaa gga aaa ccg
ttc cga tat 262 Cys Arg Val Gln Phe Asn Arg Ile Thr Glu Gly Lys Pro
Phe Arg Tyr 35 40 45 50 aaa gat cat agg aat gat gta tat tgt tct tat
ttg gga att cct tat 310 Lys Asp His Arg Asn Asp Val Tyr Cys Ser Tyr
Leu Gly Ile Pro Tyr 55 60 65 gcc gaa ccg cct att gga cca tta cga
ttt cag tct cca aaa cca ata 358 Ala Glu Pro Pro Ile Gly Pro Leu Arg
Phe Gln Ser Pro Lys Pro Ile 70 75 80 tca aat cca aaa aca gga ttc
gta cag gct cga act ttg gga gac aaa 406 Ser Asn Pro Lys Thr Gly Phe
Val Gln Ala Arg Thr Leu Gly Asp Lys 85 90 95 tgt ttc cag gaa agt
cta ata tat tct tat gca gga agc gaa gat tgc 454 Cys Phe Gln Glu Ser
Leu Ile Tyr Ser Tyr Ala Gly Ser Glu Asp Cys 100 105 110 tta tat ctg
aat ata ttc acg cca gag act gtt aat tct gcg aac aat 502 Leu Tyr Leu
Asn Ile Phe Thr Pro Glu Thr Val Asn Ser Ala Asn Asn 115 120 125 130
aca aaa tat cct gta atg ttc t 524 Thr Lys Tyr Pro Val Met Phe 135
11 137 PRT Ctenocephalides felis 11 Met Leu Pro His Ser Ser Ala Leu
Val Leu Phe Leu Phe Phe Leu Phe 1 5 10 15 Phe Leu Phe Thr Pro Ile
Leu Cys Ile Leu Trp Asp Asn Leu Asp Gln 20 25 30 His Leu Cys Arg
Val Gln Phe Asn Arg Ile Thr Glu Gly Lys Pro Phe 35 40 45 Arg Tyr
Lys Asp His Arg Asn Asp Val Tyr Cys Ser Tyr Leu Gly Ile 50 55 60
Pro Tyr Ala Glu Pro Pro Ile Gly Pro Leu Arg Phe Gln Ser Pro Lys 65
70 75 80 Pro Ile Ser Asn Pro Lys Thr Gly Phe Val Gln Ala Arg Thr
Leu Gly 85 90 95 Asp Lys Cys Phe Gln Glu Ser Leu Ile Tyr Ser Tyr
Ala Gly Ser Glu 100 105 110 Asp Cys Leu Tyr Leu Asn Ile Phe Thr Pro
Glu Thr Val Asn Ser Ala 115 120 125 Asn Asn Thr Lys Tyr Pro Val Met
Phe 130 135 12 524 DNA Ctenocephalides felis 12 agaacattac
aggatatttt gtattgttcg cagaattaac agtctctggc gtgaatatat 60
tcagatataa gcaatcttcg cttcctgcat aagaatatat tagactttcc tggaaacatt
120 tgtctcccaa agttcgagcc tgtacgaatc ctgtttttgg atttgatatt
ggttttggag 180 actgaaatcg taatggtcca ataggcggtt cggcataagg
aattcccaaa taagaacaat 240 atacatcatt cctatgatct ttatatcgga
acggttttcc ttccgtgatc ctgttaaatt 300 gaactctgca caaatgctga
tctaggttat cccatagtat gcacaagata ggtgtaaata 360 agaaaaataa
aaaaaataaa aataaaacta atgcactact gtgaggtaac attttttatt 420
gtgtttttta atgcattttg gttgcttaat tgttattatt tatctcgttt tgtttatgat
480 aaaatagacg ttttgaagac gacatgtcta tcgtatcaac gttc 524 13 1982
DNA Ctenocephalides felis CDS (3)..(1517) 13 at ttt agc tac aca ggt
gta cct tat gct aaa cct cct gtt gga gaa 47 Phe Ser Tyr Thr Gly Val
Pro Tyr Ala Lys Pro Pro Val Gly Glu 1 5 10 15 ctt aga ttt aag cct
cca cag aaa gct gag cca tgg caa ggt gtt ttc 95 Leu Arg Phe Lys Pro
Pro Gln Lys Ala Glu Pro Trp Gln Gly Val Phe 20 25 30 aac gcc aca
tta tac gga aat gtg tgt aaa tct tta aat ttc ttc ttg 143 Asn Ala Thr
Leu Tyr Gly Asn Val Cys Lys Ser Leu Asn Phe Phe Leu 35 40 45 aag
aaa att gaa gga gac gaa gac tgc ttg gta gta aac gtg tac gca 191 Lys
Lys Ile Glu Gly Asp Glu Asp Cys Leu Val Val Asn Val Tyr Ala 50 55
60 cca aaa aca act tct gat aaa aaa ctt cca gta ttt ttc tgg gtt cat
239 Pro Lys Thr Thr Ser Asp Lys Lys Leu Pro Val Phe Phe Trp Val His
65 70 75 ggt ggt ggt ttt gtg act gga tcc gga aat tta gaa ttc caa
agc cca 287 Gly Gly Gly Phe Val Thr Gly Ser Gly Asn Leu Glu Phe Gln
Ser Pro 80 85 90 95 gat tat tta gta rat ttt gat gtt att ttc gta act
ttc aat tac cga 335 Asp Tyr Leu Val Xaa Phe Asp Val Ile Phe Val Thr
Phe Asn Tyr Arg 100 105 110 ttg gga cct ctc gga ttt ctg aat ttg gag
ttg gag ggt gct cca gga 383 Leu Gly Pro Leu Gly Phe Leu Asn Leu Glu
Leu Glu Gly Ala Pro Gly 115 120 125 aat gta gga tta ttg gat cag gtg
gca gct ctg aaa tgg acc aaa gaa 431 Asn Val Gly Leu Leu Asp Gln Val
Ala Ala Leu Lys Trp Thr Lys Glu 130 135 140 aac att gag aaa ttt ggt
gga gat cca gaa aat att aca att ggt ggt 479 Asn Ile Glu Lys Phe Gly
Gly Asp Pro Glu Asn Ile Thr Ile Gly Gly 145 150 155 gtt tct gct ggt
gga gca agt gtt cat tat ctt ttg tta tct cat aca 527 Val Ser Ala Gly
Gly Ala Ser Val His Tyr Leu Leu Leu Ser His Thr 160 165 170 175 acc
act gga ctt tac aaa agg gca att gct caa agt gga agt gct ttt 575 Thr
Thr Gly Leu Tyr Lys Arg Ala Ile Ala Gln Ser Gly Ser Ala Phe 180 185
190 aat cca tgg gcc ttc caa aga cat cca gta aag cgt agt ctt caa ctt
623 Asn Pro Trp Ala Phe Gln Arg His Pro Val Lys Arg Ser Leu Gln Leu
195 200 205 gct gag ata ttg ggt cat ccc aca aac aat act caa gat gct
tta gaa 671 Ala Glu Ile Leu Gly His Pro Thr Asn Asn Thr Gln Asp Ala
Leu Glu 210 215 220 ttc tta caa aaa gcc ccc gta gac agt ctc ctg aag
aaa atg cca gct 719 Phe Leu Gln Lys Ala Pro Val Asp Ser Leu Leu Lys
Lys Met Pro Ala 225 230 235 gaa aca gaa ggt gaa ata ata gaa gag ttt
gtc ttc gta cca tca att 767 Glu Thr Glu Gly Glu Ile Ile Glu Glu Phe
Val Phe Val Pro Ser Ile 240 245 250 255 gaa aaa gtt ttc cca tcc cac
caa cct ttc ttg gaa gaa tca cca ttg 815 Glu Lys Val Phe Pro Ser His
Gln Pro Phe Leu Glu Glu Ser Pro Leu 260 265 270 gcc aga atg aaa tcc
gga tcc ttt aac aaa gta cct tta tta gtt gga 863 Ala Arg Met Lys Ser
Gly Ser Phe Asn Lys Val Pro Leu Leu Val Gly 275 280 285 ttt aac agt
gca gaa gga ctt ttg ttc aaa ttc ttc atg aaa gaa aaa 911 Phe Asn Ser
Ala Glu Gly Leu Leu Phe Lys Phe Phe Met Lys Glu Lys 290 295 300 cca
gag atg ctg aac caa gct gaa gca gat ttt gaa aga ctc gta cca 959 Pro
Glu Met Leu Asn Gln Ala Glu Ala Asp Phe Glu Arg Leu Val Pro 305 310
315 gcc gaa ttt gaa tta gtc cat gga tca gag gaa tcg aaa aaa ctt gca
1007 Ala Glu Phe Glu Leu Val His Gly Ser Glu Glu Ser Lys Lys Leu
Ala 320 325 330 335 gaa aaa atc agg aag ttt tac ttt gac gat aaa ccc
gtt cca gaa aat 1055 Glu Lys Ile Arg Lys Phe Tyr Phe Asp Asp Lys
Pro Val Pro Glu Asn 340 345 350 gaa cag aaa ttt att gac ttg ata gga
gat att tgg ttt act aga ggt 1103 Glu Gln Lys Phe Ile Asp Leu Ile
Gly Asp Ile Trp Phe Thr Arg Gly 355 360 365 gtt gac aag cat gtc aag
ttg tct gtg gag aaa caa gac gaa cca gtt 1151 Val Asp Lys His Val
Lys Leu Ser Val Glu Lys Gln Asp Glu Pro Val 370 375 380 tat tat tat
gaa tat tcc ttc tcg gaa agt cat cct gca aaa gga aca 1199 Tyr Tyr
Tyr Glu Tyr Ser Phe Ser Glu Ser His Pro Ala Lys Gly Thr 385 390 395
ttt ggt gat cat aat ctg act ggt gca tgc cat gga gaa gaa ctt gtg
1247 Phe Gly Asp His Asn Leu Thr Gly Ala Cys His Gly Glu Glu Leu
Val 400 405 410 415 aat tta ttc aaa gtc gag atg atg aag ctg gaa aaa
gat aaa cct aat 1295 Asn Leu Phe Lys Val Glu Met Met Lys Leu Glu
Lys Asp Lys Pro Asn 420 425 430 gtt cta tta aca aaa gat aga gta ctt
gcc atg tgg act aac ttc atc 1343 Val Leu Leu Thr Lys Asp Arg Val
Leu Ala Met Trp Thr Asn Phe Ile 435 440 445 aaa aat gga aat cct act
cct gaa gta aca gaa tta ttg cca gtt aaa 1391 Lys Asn Gly Asn Pro
Thr Pro Glu Val Thr Glu Leu Leu Pro
Val Lys 450 455 460 tgg gaa cct gcc aca aaa gac aag ttg aat tat ttg
aac att gat gcc 1439 Trp Glu Pro Ala Thr Lys Asp Lys Leu Asn Tyr
Leu Asn Ile Asp Ala 465 470 475 acc tta act ttg gga aca aat cct gag
gca aac cga gtc aaa ttt tgg 1487 Thr Leu Thr Leu Gly Thr Asn Pro
Glu Ala Asn Arg Val Lys Phe Trp 480 485 490 495 gaa gac gcc aca aaa
tct ttg cac ggt caa taataattta tgaaaattgt 1537 Glu Asp Ala Thr Lys
Ser Leu His Gly Gln 500 505 tttaaatact ttaggtaata tattaggtaa
ataaaaatta aaaaataaca atttttatgt 1597 tttatgtatt ggcttatgtg
tatcagttct aattttattt atttattctt gttttgcttg 1657 ttttgaaata
tcatggtttt aattttcaaa acacaacgtc gtttgttttt agcaaaattt 1717
ccaatagata tgttatatta agtactctga agtattttta tatatacact aaaatcagta
1777 aaaatacatt aactaaaaat ataagatatt ttcaataatt ttttttaaag
aaaataccaa 1837 aaataaagta aaattccaaa cggaattttt gtttaactta
aaaataaaat taactcttca 1897 ataattttga taattagtat ttctgatatc
attagtgaaa attatatttt gataatacgt 1957 atttatattt aaaataaaat tatgt
1982 14 505 PRT Ctenocephalides felis misc_feature (100)..(100) The
'Xaa' at location 100 stands for Asp, or Asn. 14 Phe Ser Tyr Thr
Gly Val Pro Tyr Ala Lys Pro Pro Val Gly Glu Leu 1 5 10 15 Arg Phe
Lys Pro Pro Gln Lys Ala Glu Pro Trp Gln Gly Val Phe Asn 20 25 30
Ala Thr Leu Tyr Gly Asn Val Cys Lys Ser Leu Asn Phe Phe Leu Lys 35
40 45 Lys Ile Glu Gly Asp Glu Asp Cys Leu Val Val Asn Val Tyr Ala
Pro 50 55 60 Lys Thr Thr Ser Asp Lys Lys Leu Pro Val Phe Phe Trp
Val His Gly 65 70 75 80 Gly Gly Phe Val Thr Gly Ser Gly Asn Leu Glu
Phe Gln Ser Pro Asp 85 90 95 Tyr Leu Val Xaa Phe Asp Val Ile Phe
Val Thr Phe Asn Tyr Arg Leu 100 105 110 Gly Pro Leu Gly Phe Leu Asn
Leu Glu Leu Glu Gly Ala Pro Gly Asn 115 120 125 Val Gly Leu Leu Asp
Gln Val Ala Ala Leu Lys Trp Thr Lys Glu Asn 130 135 140 Ile Glu Lys
Phe Gly Gly Asp Pro Glu Asn Ile Thr Ile Gly Gly Val 145 150 155 160
Ser Ala Gly Gly Ala Ser Val His Tyr Leu Leu Leu Ser His Thr Thr 165
170 175 Thr Gly Leu Tyr Lys Arg Ala Ile Ala Gln Ser Gly Ser Ala Phe
Asn 180 185 190 Pro Trp Ala Phe Gln Arg His Pro Val Lys Arg Ser Leu
Gln Leu Ala 195 200 205 Glu Ile Leu Gly His Pro Thr Asn Asn Thr Gln
Asp Ala Leu Glu Phe 210 215 220 Leu Gln Lys Ala Pro Val Asp Ser Leu
Leu Lys Lys Met Pro Ala Glu 225 230 235 240 Thr Glu Gly Glu Ile Ile
Glu Glu Phe Val Phe Val Pro Ser Ile Glu 245 250 255 Lys Val Phe Pro
Ser His Gln Pro Phe Leu Glu Glu Ser Pro Leu Ala 260 265 270 Arg Met
Lys Ser Gly Ser Phe Asn Lys Val Pro Leu Leu Val Gly Phe 275 280 285
Asn Ser Ala Glu Gly Leu Leu Phe Lys Phe Phe Met Lys Glu Lys Pro 290
295 300 Glu Met Leu Asn Gln Ala Glu Ala Asp Phe Glu Arg Leu Val Pro
Ala 305 310 315 320 Glu Phe Glu Leu Val His Gly Ser Glu Glu Ser Lys
Lys Leu Ala Glu 325 330 335 Lys Ile Arg Lys Phe Tyr Phe Asp Asp Lys
Pro Val Pro Glu Asn Glu 340 345 350 Gln Lys Phe Ile Asp Leu Ile Gly
Asp Ile Trp Phe Thr Arg Gly Val 355 360 365 Asp Lys His Val Lys Leu
Ser Val Glu Lys Gln Asp Glu Pro Val Tyr 370 375 380 Tyr Tyr Glu Tyr
Ser Phe Ser Glu Ser His Pro Ala Lys Gly Thr Phe 385 390 395 400 Gly
Asp His Asn Leu Thr Gly Ala Cys His Gly Glu Glu Leu Val Asn 405 410
415 Leu Phe Lys Val Glu Met Met Lys Leu Glu Lys Asp Lys Pro Asn Val
420 425 430 Leu Leu Thr Lys Asp Arg Val Leu Ala Met Trp Thr Asn Phe
Ile Lys 435 440 445 Asn Gly Asn Pro Thr Pro Glu Val Thr Glu Leu Leu
Pro Val Lys Trp 450 455 460 Glu Pro Ala Thr Lys Asp Lys Leu Asn Tyr
Leu Asn Ile Asp Ala Thr 465 470 475 480 Leu Thr Leu Gly Thr Asn Pro
Glu Ala Asn Arg Val Lys Phe Trp Glu 485 490 495 Asp Ala Thr Lys Ser
Leu His Gly Gln 500 505 15 1982 DNA Ctenocephalides felis 15
acataatttt attttaaata taaatacgta ttatcaaaat ataattttca ctaatgatat
60 cagaaatact aattatcaaa attattgaag agttaatttt atttttaagt
taaacaaaaa 120 ttccgtttgg aattttactt tatttttggt attttcttta
aaaaaaatta ttgaaaatat 180 cttatatttt tagttaatgt atttttactg
attttagtgt atatataaaa atacttcaga 240 gtacttaata taacatatct
attggaaatt ttgctaaaaa caaacgacgt tgtgttttga 300 aaattaaaac
catgatattt caaaacaagc aaaacaagaa taaataaata aaattagaac 360
tgatacacat aagccaatac ataaaacata aaaattgtta ttttttaatt tttatttacc
420 taatatatta cctaaagtat ttaaaacaat tttcataaat tattattgac
cgtgcaaaga 480 ttttgtggcg tcttcccaaa atttgactcg gtttgcctca
ggatttgttc ccaaagttaa 540 ggtggcatca atgttcaaat aattcaactt
gtcttttgtg gcaggttccc atttaactgg 600 caataattct gttacttcag
gagtaggatt tccatttttg atgaagttag tccacatggc 660 aagtactcta
tcttttgtta atagaacatt aggtttatct ttttccagct tcatcatctc 720
gactttgaat aaattcacaa gttcttctcc atggcatgca ccagtcagat tatgatcacc
780 aaatgttcct tttgcaggat gactttccga gaaggaatat tcataataat
aaactggttc 840 gtcttgtttc tccacagaca acttgacatg cttgtcaaca
cctctagtaa accaaatatc 900 tcctatcaag tcaataaatt tctgttcatt
ttctggaacg ggtttatcgt caaagtaaaa 960 cttcctgatt ttttctgcaa
gttttttcga ttcctctgat ccatggacta attcaaattc 1020 ggctggtacg
agtctttcaa aatctgcttc agcttggttc agcatctctg gtttttcttt 1080
catgaagaat ttgaacaaaa gtccttctgc actgttaaat ccaactaata aaggtacttt
1140 gttaaaggat ccggatttca ttctggccaa tggtgattct tccaagaaag
gttggtggga 1200 tgggaaaact ttttcaattg atggtacgaa gacaaactct
tctattattt caccttctgt 1260 ttcagctggc attttcttca ggagactgtc
tacgggggct ttttgtaaga attctaaagc 1320 atcttgagta ttgtttgtgg
gatgacccaa tatctcagca agttgaagac tacgctttac 1380 tggatgtctt
tggaaggccc atggattaaa agcacttcca ctttgagcaa ttgccctttt 1440
gtaaagtcca gtggttgtat gagataacaa aagataatga acacttgctc caccagcaga
1500 aacaccacca attgtaatat tttctggatc tccaccaaat ttctcaatgt
tttctttggt 1560 ccatttcaga gctgccacct gatccaataa tcctacattt
cctggagcac cctccaactc 1620 caaattcaga aatccgagag gtcccaatcg
gtaattgaaa gttacgaaaa taacatcaaa 1680 atytactaaa taatctgggc
tttggaattc taaatttccg gatccagtca caaaaccacc 1740 accatgaacc
cagaaaaata ctggaagttt tttatcagaa gttgtttttg gtgcgtacac 1800
gtttactacc aagcagtctt cgtctccttc aattttcttc aagaagaaat ttaaagattt
1860 acacacattt ccgtataatg tggcgttgaa aacaccttgc catggctcag
ctttctgtgg 1920 aggcttaaat ctaagttctc caacaggagg tttagcataa
ggtacacctg tgtagctaaa 1980 at 1982 16 1515 DNA Ctenocephalides
felis exon (1)..(1515) 16 ttt agc tac aca ggt gta cct tat gct aaa
cct cct gtt gga gaa ctt 48 Phe Ser Tyr Thr Gly Val Pro Tyr Ala Lys
Pro Pro Val Gly Glu Leu 1 5 10 15 aga ttt aag cct cca cag aaa gct
gag cca tgg caa ggt gtt ttc aac 96 Arg Phe Lys Pro Pro Gln Lys Ala
Glu Pro Trp Gln Gly Val Phe Asn 20 25 30 gcc aca tta tac gga aat
gtg tgt aaa tct tta aat ttc ttc ttg aag 144 Ala Thr Leu Tyr Gly Asn
Val Cys Lys Ser Leu Asn Phe Phe Leu Lys 35 40 45 aaa att gaa gga
gac gaa gac tgc ttg gta gta aac gtg tac gca cca 192 Lys Ile Glu Gly
Asp Glu Asp Cys Leu Val Val Asn Val Tyr Ala Pro 50 55 60 aaa aca
act tct gat aaa aaa ctt cca gta ttt ttc tgg gtt cat ggt 240 Lys Thr
Thr Ser Asp Lys Lys Leu Pro Val Phe Phe Trp Val His Gly 65 70 75 80
ggt ggt ttt gtg act gga tcc gga aat tta gaa ttc caa agc cca gat 288
Gly Gly Phe Val Thr Gly Ser Gly Asn Leu Glu Phe Gln Ser Pro Asp 85
90 95 tat tta gta rat ttt gat gtt att ttc gta act ttc aat tac cga
ttg 336 Tyr Leu Val Xaa Phe Asp Val Ile Phe Val Thr Phe Asn Tyr Arg
Leu 100 105 110 gga cct ctc gga ttt ctg aat ttg gag ttg gag ggt gct
cca gga aat 384 Gly Pro Leu Gly Phe Leu Asn Leu Glu Leu Glu Gly Ala
Pro Gly Asn 115 120 125 gta gga tta ttg gat cag gtg gca gct ctg aaa
tgg acc aaa gaa aac 432 Val Gly Leu Leu Asp Gln Val Ala Ala Leu Lys
Trp Thr Lys Glu Asn 130 135 140 att gag aaa ttt ggt gga gat cca gaa
aat att aca att ggt ggt gtt 480 Ile Glu Lys Phe Gly Gly Asp Pro Glu
Asn Ile Thr Ile Gly Gly Val 145 150 155 160 tct gct ggt gga gca agt
gtt cat tat ctt ttg tta tct cat aca acc 528 Ser Ala Gly Gly Ala Ser
Val His Tyr Leu Leu Leu Ser His Thr Thr 165 170 175 act gga ctt tac
aaa agg gca att gct caa agt gga agt gct ttt aat 576 Thr Gly Leu Tyr
Lys Arg Ala Ile Ala Gln Ser Gly Ser Ala Phe Asn 180 185 190 cca tgg
gcc ttc caa aga cat cca gta aag cgt agt ctt caa ctt gct 624 Pro Trp
Ala Phe Gln Arg His Pro Val Lys Arg Ser Leu Gln Leu Ala 195 200 205
gag ata ttg ggt cat ccc aca aac aat act caa gat gct tta gaa ttc 672
Glu Ile Leu Gly His Pro Thr Asn Asn Thr Gln Asp Ala Leu Glu Phe 210
215 220 tta caa aaa gcc ccc gta gac agt ctc ctg aag aaa atg cca gct
gaa 720 Leu Gln Lys Ala Pro Val Asp Ser Leu Leu Lys Lys Met Pro Ala
Glu 225 230 235 240 aca gaa ggt gaa ata ata gaa gag ttt gtc ttc gta
cca tca att gaa 768 Thr Glu Gly Glu Ile Ile Glu Glu Phe Val Phe Val
Pro Ser Ile Glu 245 250 255 aaa gtt ttc cca tcc cac caa cct ttc ttg
gaa gaa tca cca ttg gcc 816 Lys Val Phe Pro Ser His Gln Pro Phe Leu
Glu Glu Ser Pro Leu Ala 260 265 270 aga atg aaa tcc gga tcc ttt aac
aaa gta cct tta tta gtt gga ttt 864 Arg Met Lys Ser Gly Ser Phe Asn
Lys Val Pro Leu Leu Val Gly Phe 275 280 285 aac agt gca gaa gga ctt
ttg ttc aaa ttc ttc atg aaa gaa aaa cca 912 Asn Ser Ala Glu Gly Leu
Leu Phe Lys Phe Phe Met Lys Glu Lys Pro 290 295 300 gag atg ctg aac
caa gct gaa gca gat ttt gaa aga ctc gta cca gcc 960 Glu Met Leu Asn
Gln Ala Glu Ala Asp Phe Glu Arg Leu Val Pro Ala 305 310 315 320 gaa
ttt gaa tta gtc cat gga tca gag gaa tcg aaa aaa ctt gca gaa 1008
Glu Phe Glu Leu Val His Gly Ser Glu Glu Ser Lys Lys Leu Ala Glu 325
330 335 aaa atc agg aag ttt tac ttt gac gat aaa ccc gtt cca gaa aat
gaa 1056 Lys Ile Arg Lys Phe Tyr Phe Asp Asp Lys Pro Val Pro Glu
Asn Glu 340 345 350 cag aaa ttt att gac ttg ata gga gat att tgg ttt
act aga ggt gtt 1104 Gln Lys Phe Ile Asp Leu Ile Gly Asp Ile Trp
Phe Thr Arg Gly Val 355 360 365 gac aag cat gtc aag ttg tct gtg gag
aaa caa gac gaa cca gtt tat 1152 Asp Lys His Val Lys Leu Ser Val
Glu Lys Gln Asp Glu Pro Val Tyr 370 375 380 tat tat gaa tat tcc ttc
tcg gaa agt cat cct gca aaa gga aca ttt 1200 Tyr Tyr Glu Tyr Ser
Phe Ser Glu Ser His Pro Ala Lys Gly Thr Phe 385 390 395 400 ggt gat
cat aat ctg act ggt gca tgc cat gga gaa gaa ctt gtg aat 1248 Gly
Asp His Asn Leu Thr Gly Ala Cys His Gly Glu Glu Leu Val Asn 405 410
415 tta ttc aaa gtc gag atg atg aag ctg gaa aaa gat aaa cct aat gtt
1296 Leu Phe Lys Val Glu Met Met Lys Leu Glu Lys Asp Lys Pro Asn
Val 420 425 430 cta tta aca aaa gat aga gta ctt gcc atg tgg act aac
ttc atc aaa 1344 Leu Leu Thr Lys Asp Arg Val Leu Ala Met Trp Thr
Asn Phe Ile Lys 435 440 445 aat gga aat cct act cct gaa gta aca gaa
tta ttg cca gtt aaa tgg 1392 Asn Gly Asn Pro Thr Pro Glu Val Thr
Glu Leu Leu Pro Val Lys Trp 450 455 460 gaa cct gcc aca aaa gac aag
ttg aat tat ttg aac att gat gcc acc 1440 Glu Pro Ala Thr Lys Asp
Lys Leu Asn Tyr Leu Asn Ile Asp Ala Thr 465 470 475 480 tta act ttg
gga aca aat cct gag gca aac cga gtc aaa ttt tgg gaa 1488 Leu Thr
Leu Gly Thr Asn Pro Glu Ala Asn Arg Val Lys Phe Trp Glu 485 490 495
gac gcc aca aaa tct ttg cac ggt caa 1515 Asp Ala Thr Lys Ser Leu
His Gly Gln 500 505 17 1515 DNA Ctenocephalides felis 17 ttgaccgtgc
aaagattttg tggcgtcttc ccaaaatttg actcggtttg cctcaggatt 60
tgttcccaaa gttaaggtgg catcaatgtt caaataattc aacttgtctt ttgtggcagg
120 ttcccattta actggcaata attctgttac ttcaggagta ggatttccat
ttttgatgaa 180 gttagtccac atggcaagta ctctatcttt tgttaataga
acattaggtt tatctttttc 240 cagcttcatc atctcgactt tgaataaatt
cacaagttct tctccatggc atgcaccagt 300 cagattatga tcaccaaatg
ttccttttgc aggatgactt tccgagaagg aatattcata 360 ataataaact
ggttcgtctt gtttctccac agacaacttg acatgcttgt caacacctct 420
agtaaaccaa atatctccta tcaagtcaat aaatttctgt tcattttctg gaacgggttt
480 atcgtcaaag taaaacttcc tgattttttc tgcaagtttt ttcgattcct
ctgatccatg 540 gactaattca aattcggctg gtacgagtct ttcaaaatct
gcttcagctt ggttcagcat 600 ctctggtttt tctttcatga agaatttgaa
caaaagtcct tctgcactgt taaatccaac 660 taataaaggt actttgttaa
aggatccgga tttcattctg gccaatggtg attcttccaa 720 gaaaggttgg
tgggatggga aaactttttc aattgatggt acgaagacaa actcttctat 780
tatttcacct tctgtttcag ctggcatttt cttcaggaga ctgtctacgg gggctttttg
840 taagaattct aaagcatctt gagtattgtt tgtgggatga cccaatatct
cagcaagttg 900 aagactacgc tttactggat gtctttggaa ggcccatgga
ttaaaagcac ttccactttg 960 agcaattgcc cttttgtaaa gtccagtggt
tgtatgagat aacaaaagat aatgaacact 1020 tgctccacca gcagaaacac
caccaattgt aatattttct ggatctccac caaatttctc 1080 aatgttttct
ttggtccatt tcagagctgc cacctgatcc aataatccta catttcctgg 1140
agcaccctcc aactccaaat tcagaaatcc gagaggtccc aatcggtaat tgaaagttac
1200 gaaaataaca tcaaaatyta ctaaataatc tgggctttgg aattctaaat
ttccggatcc 1260 agtcacaaaa ccaccaccat gaacccagaa aaatactgga
agttttttat cagaagttgt 1320 ttttggtgcg tacacgttta ctaccaagca
gtcttcgtct ccttcaattt tcttcaagaa 1380 gaaatttaaa gatttacaca
catttccgta taatgtggcg ttgaaaacac cttgccatgg 1440 ctcagctttc
tgtggaggct taaatctaag ttctccaaca ggaggtttag cataaggtac 1500
acctgtgtag ctaaa 1515 18 1792 DNA Ctenocephalides felis CDS
(49)..(1701) 18 actgtgtgct aataattcag tacacacagt caatagtcta
gatccaag atg tct cgt 57 Met Ser Arg 1 gtt att ttt tta agt tgt att
ttt ttg ttt agt ttt aat ttt ata aaa 105 Val Ile Phe Leu Ser Cys Ile
Phe Leu Phe Ser Phe Asn Phe Ile Lys 5 10 15 tgt gat ccc ccg act gta
act ttg ccc cag ggc gaa ttg gtt gga aaa 153 Cys Asp Pro Pro Thr Val
Thr Leu Pro Gln Gly Glu Leu Val Gly Lys 20 25 30 35 gct ttg acg aac
gaa aat gga aaa gag tat ttt agc tac aca ggt gtg 201 Ala Leu Thr Asn
Glu Asn Gly Lys Glu Tyr Phe Ser Tyr Thr Gly Val 40 45 50 cct tat
gct aaa cct cca gtt gga gaa ctt aga ttt aag cct cca cag 249 Pro Tyr
Ala Lys Pro Pro Val Gly Glu Leu Arg Phe Lys Pro Pro Gln 55 60 65
aaa gct gag cca tgg aat ggt gtt ttc aac gcc aca tca cat gga aat 297
Lys Ala Glu Pro Trp Asn Gly Val Phe Asn Ala Thr Ser His Gly Asn 70
75 80 gtg tgc aaa gct ttg aat ttc ttc ttg aaa aaa att gaa gga gac
gaa 345 Val Cys Lys Ala Leu Asn Phe Phe Leu Lys Lys Ile Glu Gly Asp
Glu 85 90 95 gac tgc ttg ttg gtg aat gtg tac gca cca aaa aca act
tct gac aaa 393 Asp Cys Leu Leu Val Asn Val Tyr Ala Pro Lys Thr Thr
Ser Asp Lys 100 105 110 115 aaa ctt cca gta ttt ttc tgg gtt cat ggt
ggc ggt ttt gtg act gga 441 Lys Leu Pro Val Phe Phe Trp Val His Gly
Gly Gly Phe Val Thr Gly 120 125 130 tcc gga aat tta gaa ttt caa agc
cca gat tat tta gta aat tat gat 489 Ser Gly Asn Leu Glu Phe Gln Ser
Pro Asp Tyr Leu Val Asn Tyr Asp 135 140 145 gtt att ttt gta act ttc
aat tac cga ttg gga cca ctc gga ttt ttg 537 Val Ile Phe Val Thr Phe
Asn Tyr Arg Leu Gly Pro Leu Gly Phe Leu 150 155 160 aat ttg gag ttg
gaa ggt gct cct gga aat gta gga tta ttg gat cag 585 Asn Leu Glu Leu
Glu Gly Ala Pro Gly Asn Val Gly Leu Leu Asp Gln 165 170 175 gta gca
gct ttg aaa tgg acc aaa gaa aat att gag aaa ttt ggt gga 633 Val Ala
Ala Leu Lys Trp Thr Lys Glu Asn Ile Glu Lys Phe Gly Gly 180 185
190
195 gat cca gaa aat att aca att ggt ggt gtt tct gct ggt gga gca agt
681 Asp Pro Glu Asn Ile Thr Ile Gly Gly Val Ser Ala Gly Gly Ala Ser
200 205 210 gtt cat tat ctt tta ttg tca cat aca acc act gga ctt tac
aaa agg 729 Val His Tyr Leu Leu Leu Ser His Thr Thr Thr Gly Leu Tyr
Lys Arg 215 220 225 gca att gct caa agt gga agt gct tta aat cca tgg
gcc ttc caa aga 777 Ala Ile Ala Gln Ser Gly Ser Ala Leu Asn Pro Trp
Ala Phe Gln Arg 230 235 240 cat cca gta aag cgt agt ctt caa ctt gct
gag ata tta ggt cat ccc 825 His Pro Val Lys Arg Ser Leu Gln Leu Ala
Glu Ile Leu Gly His Pro 245 250 255 aca aac aac act caa gat gct tta
gaa ttc tta caa aaa gcc cca gta 873 Thr Asn Asn Thr Gln Asp Ala Leu
Glu Phe Leu Gln Lys Ala Pro Val 260 265 270 275 gac agt ctc ctg aaa
aaa atg cca gct gaa aca gaa ggt gaa ata ata 921 Asp Ser Leu Leu Lys
Lys Met Pro Ala Glu Thr Glu Gly Glu Ile Ile 280 285 290 gaa gag ttc
gtc ttc gta cca tca att gaa aaa gtt ttc cca tcc cac 969 Glu Glu Phe
Val Phe Val Pro Ser Ile Glu Lys Val Phe Pro Ser His 295 300 305 caa
cct ttc ttg gaa gaa tca cca ttg gcc aga atg aaa tct gga tcc 1017
Gln Pro Phe Leu Glu Glu Ser Pro Leu Ala Arg Met Lys Ser Gly Ser 310
315 320 ttt aac aaa gta cct tta tta gtt gga ttc aac agc gca gaa gga
ctt 1065 Phe Asn Lys Val Pro Leu Leu Val Gly Phe Asn Ser Ala Glu
Gly Leu 325 330 335 ttg tac aaa ttc ttt atg aaa gaa aaa cca gag atg
ctg aac caa gct 1113 Leu Tyr Lys Phe Phe Met Lys Glu Lys Pro Glu
Met Leu Asn Gln Ala 340 345 350 355 gaa gca gat ttc gaa aga ctc gta
cca gcc gaa ttt gaa tta gcc cat 1161 Glu Ala Asp Phe Glu Arg Leu
Val Pro Ala Glu Phe Glu Leu Ala His 360 365 370 gga tca gaa gaa tcg
aaa aaa ctt gca gaa aaa atc agg aag ttt tac 1209 Gly Ser Glu Glu
Ser Lys Lys Leu Ala Glu Lys Ile Arg Lys Phe Tyr 375 380 385 ttt gac
gat aaa ccc gtt cct gaa aat gag cag aaa ttt att gac ttg 1257 Phe
Asp Asp Lys Pro Val Pro Glu Asn Glu Gln Lys Phe Ile Asp Leu 390 395
400 ata gga gat att tgg ttt act aga ggc att gac aag cat gtc aag ttg
1305 Ile Gly Asp Ile Trp Phe Thr Arg Gly Ile Asp Lys His Val Lys
Leu 405 410 415 tct gta gaa aaa caa gac gag cca gta tat tat tat gaa
tat tct ttc 1353 Ser Val Glu Lys Gln Asp Glu Pro Val Tyr Tyr Tyr
Glu Tyr Ser Phe 420 425 430 435 tct gaa agt cat cct gca aaa gga aca
ttt ggt gac cat aac ttg act 1401 Ser Glu Ser His Pro Ala Lys Gly
Thr Phe Gly Asp His Asn Leu Thr 440 445 450 gga gca tgt cat ggt gaa
gaa ctt gtg aat tta ttc aaa gtc gag atg 1449 Gly Ala Cys His Gly
Glu Glu Leu Val Asn Leu Phe Lys Val Glu Met 455 460 465 atg aag ctg
gaa aaa gat aaa ccg aat gtt tta tta aca aaa gat agg 1497 Met Lys
Leu Glu Lys Asp Lys Pro Asn Val Leu Leu Thr Lys Asp Arg 470 475 480
gta ctt gct atg tgg acg aac ttc atc aaa aat gga aat cct act cct
1545 Val Leu Ala Met Trp Thr Asn Phe Ile Lys Asn Gly Asn Pro Thr
Pro 485 490 495 gaa gta act gaa tta ttg cca gtt aaa tgg gaa cct gcc
aca aaa gac 1593 Glu Val Thr Glu Leu Leu Pro Val Lys Trp Glu Pro
Ala Thr Lys Asp 500 505 510 515 aag ttg aat tat ttg aac att gat gcc
acc tta act ttg gga aca aat 1641 Lys Leu Asn Tyr Leu Asn Ile Asp
Ala Thr Leu Thr Leu Gly Thr Asn 520 525 530 cca gaa gaa acc cga gtc
aaa tty tgg gaa gat gcc aca aaa act ttg 1689 Pro Glu Glu Thr Arg
Val Lys Phe Trp Glu Asp Ala Thr Lys Thr Leu 535 540 545 cac agt caa
taa aaatgtatga aaattgtttt aattatttta ggtaatacat 1741 His Ser Gln
550 taggtaaata aaaattnaaa aataacnaaa aaaaaaaaaa aaaaaaaaaa a 1792
19 550 PRT Ctenocephalides felis 19 Met Ser Arg Val Ile Phe Leu Ser
Cys Ile Phe Leu Phe Ser Phe Asn 1 5 10 15 Phe Ile Lys Cys Asp Pro
Pro Thr Val Thr Leu Pro Gln Gly Glu Leu 20 25 30 Val Gly Lys Ala
Leu Thr Asn Glu Asn Gly Lys Glu Tyr Phe Ser Tyr 35 40 45 Thr Gly
Val Pro Tyr Ala Lys Pro Pro Val Gly Glu Leu Arg Phe Lys 50 55 60
Pro Pro Gln Lys Ala Glu Pro Trp Asn Gly Val Phe Asn Ala Thr Ser 65
70 75 80 His Gly Asn Val Cys Lys Ala Leu Asn Phe Phe Leu Lys Lys
Ile Glu 85 90 95 Gly Asp Glu Asp Cys Leu Leu Val Asn Val Tyr Ala
Pro Lys Thr Thr 100 105 110 Ser Asp Lys Lys Leu Pro Val Phe Phe Trp
Val His Gly Gly Gly Phe 115 120 125 Val Thr Gly Ser Gly Asn Leu Glu
Phe Gln Ser Pro Asp Tyr Leu Val 130 135 140 Asn Tyr Asp Val Ile Phe
Val Thr Phe Asn Tyr Arg Leu Gly Pro Leu 145 150 155 160 Gly Phe Leu
Asn Leu Glu Leu Glu Gly Ala Pro Gly Asn Val Gly Leu 165 170 175 Leu
Asp Gln Val Ala Ala Leu Lys Trp Thr Lys Glu Asn Ile Glu Lys 180 185
190 Phe Gly Gly Asp Pro Glu Asn Ile Thr Ile Gly Gly Val Ser Ala Gly
195 200 205 Gly Ala Ser Val His Tyr Leu Leu Leu Ser His Thr Thr Thr
Gly Leu 210 215 220 Tyr Lys Arg Ala Ile Ala Gln Ser Gly Ser Ala Leu
Asn Pro Trp Ala 225 230 235 240 Phe Gln Arg His Pro Val Lys Arg Ser
Leu Gln Leu Ala Glu Ile Leu 245 250 255 Gly His Pro Thr Asn Asn Thr
Gln Asp Ala Leu Glu Phe Leu Gln Lys 260 265 270 Ala Pro Val Asp Ser
Leu Leu Lys Lys Met Pro Ala Glu Thr Glu Gly 275 280 285 Glu Ile Ile
Glu Glu Phe Val Phe Val Pro Ser Ile Glu Lys Val Phe 290 295 300 Pro
Ser His Gln Pro Phe Leu Glu Glu Ser Pro Leu Ala Arg Met Lys 305 310
315 320 Ser Gly Ser Phe Asn Lys Val Pro Leu Leu Val Gly Phe Asn Ser
Ala 325 330 335 Glu Gly Leu Leu Tyr Lys Phe Phe Met Lys Glu Lys Pro
Glu Met Leu 340 345 350 Asn Gln Ala Glu Ala Asp Phe Glu Arg Leu Val
Pro Ala Glu Phe Glu 355 360 365 Leu Ala His Gly Ser Glu Glu Ser Lys
Lys Leu Ala Glu Lys Ile Arg 370 375 380 Lys Phe Tyr Phe Asp Asp Lys
Pro Val Pro Glu Asn Glu Gln Lys Phe 385 390 395 400 Ile Asp Leu Ile
Gly Asp Ile Trp Phe Thr Arg Gly Ile Asp Lys His 405 410 415 Val Lys
Leu Ser Val Glu Lys Gln Asp Glu Pro Val Tyr Tyr Tyr Glu 420 425 430
Tyr Ser Phe Ser Glu Ser His Pro Ala Lys Gly Thr Phe Gly Asp His 435
440 445 Asn Leu Thr Gly Ala Cys His Gly Glu Glu Leu Val Asn Leu Phe
Lys 450 455 460 Val Glu Met Met Lys Leu Glu Lys Asp Lys Pro Asn Val
Leu Leu Thr 465 470 475 480 Lys Asp Arg Val Leu Ala Met Trp Thr Asn
Phe Ile Lys Asn Gly Asn 485 490 495 Pro Thr Pro Glu Val Thr Glu Leu
Leu Pro Val Lys Trp Glu Pro Ala 500 505 510 Thr Lys Asp Lys Leu Asn
Tyr Leu Asn Ile Asp Ala Thr Leu Thr Leu 515 520 525 Gly Thr Asn Pro
Glu Glu Thr Arg Val Lys Phe Trp Glu Asp Ala Thr 530 535 540 Lys Thr
Leu His Ser Gln 545 550 20 1792 DNA Ctenocephalides felis
misc_feature (25)..(25) n = unknown 20 tttttttttt tttttttttt
ttttngttat ttttnaattt ttatttacct aatgtattac 60 ctaaaataat
taaaacaatt ttcatacatt tttattgact gtgcaaagtt tttgtggcat 120
cttcccaraa tttgactcgg gtttcttctg gatttgttcc caaagttaag gtggcatcaa
180 tgttcaaata attcaacttg tcttttgtgg caggttccca tttaactggc
aataattcag 240 ttacttcagg agtaggattt ccatttttga tgaagttcgt
ccacatagca agtaccctat 300 cttttgttaa taaaacattc ggtttatctt
tttccagctt catcatctcg actttgaata 360 aattcacaag ttcttcacca
tgacatgctc cagtcaagtt atggtcacca aatgttcctt 420 ttgcaggatg
actttcagag aaagaatatt cataataata tactggctcg tcttgttttt 480
ctacagacaa cttgacatgc ttgtcaatgc ctctagtaaa ccaaatatct cctatcaagt
540 caataaattt ctgctcattt tcaggaacgg gtttatcgtc aaagtaaaac
ttcctgattt 600 tttctgcaag ttttttcgat tcttctgatc catgggctaa
ttcaaattcg gctggtacga 660 gtctttcgaa atctgcttca gcttggttca
gcatctctgg tttttctttc ataaagaatt 720 tgtacaaaag tccttctgcg
ctgttgaatc caactaataa aggtactttg ttaaaggatc 780 cagatttcat
tctggccaat ggtgattctt ccaagaaagg ttggtgggat gggaaaactt 840
tttcaattga tggtacgaag acgaactctt ctattatttc accttctgtt tcagctggca
900 tttttttcag gagactgtct actggggctt tttgtaagaa ttctaaagca
tcttgagtgt 960 tgtttgtggg atgacctaat atctcagcaa gttgaagact
acgctttact ggatgtcttt 1020 ggaaggccca tggatttaaa gcacttccac
tttgagcaat tgcccttttg taaagtccag 1080 tggttgtatg tgacaataaa
agataatgaa cacttgctcc accagcagaa acaccaccaa 1140 ttgtaatatt
ttctggatct ccaccaaatt tctcaatatt ttctttggtc catttcaaag 1200
ctgctacctg atccaataat cctacatttc caggagcacc ttccaactcc aaattcaaaa
1260 atccgagtgg tcccaatcgg taattgaaag ttacaaaaat aacatcataa
tttactaaat 1320 aatctgggct ttgaaattct aaatttccgg atccagtcac
aaaaccgcca ccatgaaccc 1380 agaaaaatac tggaagtttt ttgtcagaag
ttgtttttgg tgcgtacaca ttcaccaaca 1440 agcagtcttc gtctccttca
atttttttca agaagaaatt caaagctttg cacacatttc 1500 catgtgatgt
ggcgttgaaa acaccattcc atggctcagc tttctgtgga ggcttaaatc 1560
taagttctcc aactggaggt ttagcataag gcacacctgt gtagctaaaa tactcttttc
1620 cattttcgtt cgtcaaagct tttccaacca attcgccctg gggcaaagtt
acagtcgggg 1680 gatcacattt tataaaatta aaactaaaca aaaaaataca
acttaaaaaa ataacacgag 1740 acatcttgga tctagactat tgactgtgtg
tactgaatta ttagcacaca gt 1792 21 1650 DNA Ctenocephalides felis
exon (1)..(1650) 21 atg tct cgt gtt att ttt tta agt tgt att ttt ttg
ttt agt ttt aat 48 Met Ser Arg Val Ile Phe Leu Ser Cys Ile Phe Leu
Phe Ser Phe Asn 1 5 10 15 ttt ata aaa tgt gat ccc ccg act gta act
ttg ccc cag ggc gaa ttg 96 Phe Ile Lys Cys Asp Pro Pro Thr Val Thr
Leu Pro Gln Gly Glu Leu 20 25 30 gtt gga aaa gct ttg acg aac gaa
aat gga aaa gag tat ttt agc tac 144 Val Gly Lys Ala Leu Thr Asn Glu
Asn Gly Lys Glu Tyr Phe Ser Tyr 35 40 45 aca ggt gtg cct tat gct
aaa cct cca gtt gga gaa ctt aga ttt aag 192 Thr Gly Val Pro Tyr Ala
Lys Pro Pro Val Gly Glu Leu Arg Phe Lys 50 55 60 cct cca cag aaa
gct gag cca tgg aat ggt gtt ttc aac gcc aca tca 240 Pro Pro Gln Lys
Ala Glu Pro Trp Asn Gly Val Phe Asn Ala Thr Ser 65 70 75 80 cat gga
aat gtg tgc aaa gct ttg aat ttc ttc ttg aaa aaa att gaa 288 His Gly
Asn Val Cys Lys Ala Leu Asn Phe Phe Leu Lys Lys Ile Glu 85 90 95
gga gac gaa gac tgc ttg ttg gtg aat gtg tac gca cca aaa aca act 336
Gly Asp Glu Asp Cys Leu Leu Val Asn Val Tyr Ala Pro Lys Thr Thr 100
105 110 tct gac aaa aaa ctt cca gta ttt ttc tgg gtt cat ggt ggc ggt
ttt 384 Ser Asp Lys Lys Leu Pro Val Phe Phe Trp Val His Gly Gly Gly
Phe 115 120 125 gtg act gga tcc gga aat tta gaa ttt caa agc cca gat
tat tta gta 432 Val Thr Gly Ser Gly Asn Leu Glu Phe Gln Ser Pro Asp
Tyr Leu Val 130 135 140 aat tat gat gtt att ttt gta act ttc aat tac
cga ttg gga cca ctc 480 Asn Tyr Asp Val Ile Phe Val Thr Phe Asn Tyr
Arg Leu Gly Pro Leu 145 150 155 160 gga ttt ttg aat ttg gag ttg gaa
ggt gct cct gga aat gta gga tta 528 Gly Phe Leu Asn Leu Glu Leu Glu
Gly Ala Pro Gly Asn Val Gly Leu 165 170 175 ttg gat cag gta gca gct
ttg aaa tgg acc aaa gaa aat att gag aaa 576 Leu Asp Gln Val Ala Ala
Leu Lys Trp Thr Lys Glu Asn Ile Glu Lys 180 185 190 ttt ggt gga gat
cca gaa aat att aca att ggt ggt gtt tct gct ggt 624 Phe Gly Gly Asp
Pro Glu Asn Ile Thr Ile Gly Gly Val Ser Ala Gly 195 200 205 gga gca
agt gtt cat tat ctt tta ttg tca cat aca acc act gga ctt 672 Gly Ala
Ser Val His Tyr Leu Leu Leu Ser His Thr Thr Thr Gly Leu 210 215 220
tac aaa agg gca att gct caa agt gga agt gct tta aat cca tgg gcc 720
Tyr Lys Arg Ala Ile Ala Gln Ser Gly Ser Ala Leu Asn Pro Trp Ala 225
230 235 240 ttc caa aga cat cca gta aag cgt agt ctt caa ctt gct gag
ata tta 768 Phe Gln Arg His Pro Val Lys Arg Ser Leu Gln Leu Ala Glu
Ile Leu 245 250 255 ggt cat ccc aca aac aac act caa gat gct tta gaa
ttc tta caa aaa 816 Gly His Pro Thr Asn Asn Thr Gln Asp Ala Leu Glu
Phe Leu Gln Lys 260 265 270 gcc cca gta gac agt ctc ctg aaa aaa atg
cca gct gaa aca gaa ggt 864 Ala Pro Val Asp Ser Leu Leu Lys Lys Met
Pro Ala Glu Thr Glu Gly 275 280 285 gaa ata ata gaa gag ttc gtc ttc
gta cca tca att gaa aaa gtt ttc 912 Glu Ile Ile Glu Glu Phe Val Phe
Val Pro Ser Ile Glu Lys Val Phe 290 295 300 cca tcc cac caa cct ttc
ttg gaa gaa tca cca ttg gcc aga atg aaa 960 Pro Ser His Gln Pro Phe
Leu Glu Glu Ser Pro Leu Ala Arg Met Lys 305 310 315 320 tct gga tcc
ttt aac aaa gta cct tta tta gtt gga ttc aac agc gca 1008 Ser Gly
Ser Phe Asn Lys Val Pro Leu Leu Val Gly Phe Asn Ser Ala 325 330 335
gaa gga ctt ttg tac aaa ttc ttt atg aaa gaa aaa cca gag atg ctg
1056 Glu Gly Leu Leu Tyr Lys Phe Phe Met Lys Glu Lys Pro Glu Met
Leu 340 345 350 aac caa gct gaa gca gat ttc gaa aga ctc gta cca gcc
gaa ttt gaa 1104 Asn Gln Ala Glu Ala Asp Phe Glu Arg Leu Val Pro
Ala Glu Phe Glu 355 360 365 tta gcc cat gga tca gaa gaa tcg aaa aaa
ctt gca gaa aaa atc agg 1152 Leu Ala His Gly Ser Glu Glu Ser Lys
Lys Leu Ala Glu Lys Ile Arg 370 375 380 aag ttt tac ttt gac gat aaa
ccc gtt cct gaa aat gag cag aaa ttt 1200 Lys Phe Tyr Phe Asp Asp
Lys Pro Val Pro Glu Asn Glu Gln Lys Phe 385 390 395 400 att gac ttg
ata gga gat att tgg ttt act aga ggc att gac aag cat 1248 Ile Asp
Leu Ile Gly Asp Ile Trp Phe Thr Arg Gly Ile Asp Lys His 405 410 415
gtc aag ttg tct gta gaa aaa caa gac gag cca gta tat tat tat gaa
1296 Val Lys Leu Ser Val Glu Lys Gln Asp Glu Pro Val Tyr Tyr Tyr
Glu 420 425 430 tat tct ttc tct gaa agt cat cct gca aaa gga aca ttt
ggt gac cat 1344 Tyr Ser Phe Ser Glu Ser His Pro Ala Lys Gly Thr
Phe Gly Asp His 435 440 445 aac ttg act gga gca tgt cat ggt gaa gaa
ctt gtg aat tta ttc aaa 1392 Asn Leu Thr Gly Ala Cys His Gly Glu
Glu Leu Val Asn Leu Phe Lys 450 455 460 gtc gag atg atg aag ctg gaa
aaa gat aaa ccg aat gtt tta tta aca 1440 Val Glu Met Met Lys Leu
Glu Lys Asp Lys Pro Asn Val Leu Leu Thr 465 470 475 480 aaa gat agg
gta ctt gct atg tgg acg aac ttc atc aaa aat gga aat 1488 Lys Asp
Arg Val Leu Ala Met Trp Thr Asn Phe Ile Lys Asn Gly Asn 485 490 495
cct act cct gaa gta act gaa tta ttg cca gtt aaa tgg gaa cct gcc
1536 Pro Thr Pro Glu Val Thr Glu Leu Leu Pro Val Lys Trp Glu Pro
Ala 500 505 510 aca aaa gac aag ttg aat tat ttg aac att gat gcc acc
tta act ttg 1584 Thr Lys Asp Lys Leu Asn Tyr Leu Asn Ile Asp Ala
Thr Leu Thr Leu 515 520 525 gga aca aat cca gaa gaa acc cga gtc aaa
tty tgg gaa gat gcc aca 1632 Gly Thr Asn Pro Glu Glu Thr Arg Val
Lys Phe Trp Glu Asp Ala Thr 530 535 540 aaa act ttg cac agt caa
1650 Lys Thr Leu His Ser Gln 545 550 22 1650 DNA Ctenocephalides
felis 22 ttgactgtgc aaagtttttg tggcatcttc ccaraatttg actcgggttt
cttctggatt 60 tgttcccaaa gttaaggtgg catcaatgtt caaataattc
aacttgtctt ttgtggcagg 120 ttcccattta actggcaata attcagttac
ttcaggagta ggatttccat ttttgatgaa 180 gttcgtccac atagcaagta
ccctatcttt tgttaataaa acattcggtt tatctttttc 240 cagcttcatc
atctcgactt tgaataaatt cacaagttct tcaccatgac atgctccagt 300
caagttatgg tcaccaaatg ttccttttgc aggatgactt tcagagaaag aatattcata
360 ataatatact ggctcgtctt gtttttctac agacaacttg acatgcttgt
caatgcctct 420 agtaaaccaa atatctccta tcaagtcaat aaatttctgc
tcattttcag gaacgggttt 480 atcgtcaaag taaaacttcc tgattttttc
tgcaagtttt ttcgattctt ctgatccatg 540 ggctaattca aattcggctg
gtacgagtct ttcgaaatct gcttcagctt ggttcagcat
600 ctctggtttt tctttcataa agaatttgta caaaagtcct tctgcgctgt
tgaatccaac 660 taataaaggt actttgttaa aggatccaga tttcattctg
gccaatggtg attcttccaa 720 gaaaggttgg tgggatggga aaactttttc
aattgatggt acgaagacga actcttctat 780 tatttcacct tctgtttcag
ctggcatttt tttcaggaga ctgtctactg gggctttttg 840 taagaattct
aaagcatctt gagtgttgtt tgtgggatga cctaatatct cagcaagttg 900
aagactacgc tttactggat gtctttggaa ggcccatgga tttaaagcac ttccactttg
960 agcaattgcc cttttgtaaa gtccagtggt tgtatgtgac aataaaagat
aatgaacact 1020 tgctccacca gcagaaacac caccaattgt aatattttct
ggatctccac caaatttctc 1080 aatattttct ttggtccatt tcaaagctgc
tacctgatcc aataatccta catttccagg 1140 agcaccttcc aactccaaat
tcaaaaatcc gagtggtccc aatcggtaat tgaaagttac 1200 aaaaataaca
tcataattta ctaaataatc tgggctttga aattctaaat ttccggatcc 1260
agtcacaaaa ccgccaccat gaacccagaa aaatactgga agttttttgt cagaagttgt
1320 ttttggtgcg tacacattca ccaacaagca gtcttcgtct ccttcaattt
ttttcaagaa 1380 gaaattcaaa gctttgcaca catttccatg tgatgtggcg
ttgaaaacac cattccatgg 1440 ctcagctttc tgtggaggct taaatctaag
ttctccaact ggaggtttag cataaggcac 1500 acctgtgtag ctaaaatact
cttttccatt ttcgttcgtc aaagcttttc caaccaattc 1560 gccctggggc
aaagttacag tcgggggatc acattttata aaattaaaac taaacaaaaa 1620
aatacaactt aaaaaaataa cacgagacat 1650 23 1590 DNA Ctenocephalides
felis exon (1)..(1590) 23 gat ccc ccg act gta act ttg ccc cag ggc
gaa ttg gtt gga aaa gct 48 Asp Pro Pro Thr Val Thr Leu Pro Gln Gly
Glu Leu Val Gly Lys Ala 1 5 10 15 ttg acg aac gaa aat gga aaa gag
tat ttt agc tac aca ggt gtg cct 96 Leu Thr Asn Glu Asn Gly Lys Glu
Tyr Phe Ser Tyr Thr Gly Val Pro 20 25 30 tat gct aaa cct cca gtt
gga gaa ctt aga ttt aag cct cca cag aaa 144 Tyr Ala Lys Pro Pro Val
Gly Glu Leu Arg Phe Lys Pro Pro Gln Lys 35 40 45 gct gag cca tgg
aat ggt gtt ttc aac gcc aca tca cat gga aat gtg 192 Ala Glu Pro Trp
Asn Gly Val Phe Asn Ala Thr Ser His Gly Asn Val 50 55 60 tgc aaa
gct ttg aat ttc ttc ttg aaa aaa att gaa gga gac gaa gac 240 Cys Lys
Ala Leu Asn Phe Phe Leu Lys Lys Ile Glu Gly Asp Glu Asp 65 70 75 80
tgc ttg ttg gtg aat gtg tac gca cca aaa aca act tct gac aaa aaa 288
Cys Leu Leu Val Asn Val Tyr Ala Pro Lys Thr Thr Ser Asp Lys Lys 85
90 95 ctt cca gta ttt ttc tgg gtt cat ggt ggc ggt ttt gtg act gga
tcc 336 Leu Pro Val Phe Phe Trp Val His Gly Gly Gly Phe Val Thr Gly
Ser 100 105 110 gga aat tta gaa ttt caa agc cca gat tat tta gta aat
tat gat gtt 384 Gly Asn Leu Glu Phe Gln Ser Pro Asp Tyr Leu Val Asn
Tyr Asp Val 115 120 125 att ttt gta act ttc aat tac cga ttg gga cca
ctc gga ttt ttg aat 432 Ile Phe Val Thr Phe Asn Tyr Arg Leu Gly Pro
Leu Gly Phe Leu Asn 130 135 140 ttg gag ttg gaa ggt gct cct gga aat
gta gga tta ttg gat cag gta 480 Leu Glu Leu Glu Gly Ala Pro Gly Asn
Val Gly Leu Leu Asp Gln Val 145 150 155 160 gca gct ttg aaa tgg acc
aaa gaa aat att gag aaa ttt ggt gga gat 528 Ala Ala Leu Lys Trp Thr
Lys Glu Asn Ile Glu Lys Phe Gly Gly Asp 165 170 175 cca gaa aat att
aca att ggt ggt gtt tct gct ggt gga gca agt gtt 576 Pro Glu Asn Ile
Thr Ile Gly Gly Val Ser Ala Gly Gly Ala Ser Val 180 185 190 cat tat
ctt tta ttg tca cat aca acc act gga ctt tac aaa agg gca 624 His Tyr
Leu Leu Leu Ser His Thr Thr Thr Gly Leu Tyr Lys Arg Ala 195 200 205
att gct caa agt gga agt gct tta aat cca tgg gcc ttc caa aga cat 672
Ile Ala Gln Ser Gly Ser Ala Leu Asn Pro Trp Ala Phe Gln Arg His 210
215 220 cca gta aag cgt agt ctt caa ctt gct gag ata tta ggt cat ccc
aca 720 Pro Val Lys Arg Ser Leu Gln Leu Ala Glu Ile Leu Gly His Pro
Thr 225 230 235 240 aac aac act caa gat gct tta gaa ttc tta caa aaa
gcc cca gta gac 768 Asn Asn Thr Gln Asp Ala Leu Glu Phe Leu Gln Lys
Ala Pro Val Asp 245 250 255 agt ctc ctg aaa aaa atg cca gct gaa aca
gaa ggt gaa ata ata gaa 816 Ser Leu Leu Lys Lys Met Pro Ala Glu Thr
Glu Gly Glu Ile Ile Glu 260 265 270 gag ttc gtc ttc gta cca tca att
gaa aaa gtt ttc cca tcc cac caa 864 Glu Phe Val Phe Val Pro Ser Ile
Glu Lys Val Phe Pro Ser His Gln 275 280 285 cct ttc ttg gaa gaa tca
cca ttg gcc aga atg aaa tct gga tcc ttt 912 Pro Phe Leu Glu Glu Ser
Pro Leu Ala Arg Met Lys Ser Gly Ser Phe 290 295 300 aac aaa gta cct
tta tta gtt gga ttc aac agc gca gaa gga ctt ttg 960 Asn Lys Val Pro
Leu Leu Val Gly Phe Asn Ser Ala Glu Gly Leu Leu 305 310 315 320 tac
aaa ttc ttt atg aaa gaa aaa cca gag atg ctg aac caa gct gaa 1008
Tyr Lys Phe Phe Met Lys Glu Lys Pro Glu Met Leu Asn Gln Ala Glu 325
330 335 gca gat ttc gaa aga ctc gta cca gcc gaa ttt gaa tta gcc cat
gga 1056 Ala Asp Phe Glu Arg Leu Val Pro Ala Glu Phe Glu Leu Ala
His Gly 340 345 350 tca gaa gaa tcg aaa aaa ctt gca gaa aaa atc agg
aag ttt tac ttt 1104 Ser Glu Glu Ser Lys Lys Leu Ala Glu Lys Ile
Arg Lys Phe Tyr Phe 355 360 365 gac gat aaa ccc gtt cct gaa aat gag
cag aaa ttt att gac ttg ata 1152 Asp Asp Lys Pro Val Pro Glu Asn
Glu Gln Lys Phe Ile Asp Leu Ile 370 375 380 gga gat att tgg ttt act
aga ggc att gac aag cat gtc aag ttg tct 1200 Gly Asp Ile Trp Phe
Thr Arg Gly Ile Asp Lys His Val Lys Leu Ser 385 390 395 400 gta gaa
aaa caa gac gag cca gta tat tat tat gaa tat tct ttc tct 1248 Val
Glu Lys Gln Asp Glu Pro Val Tyr Tyr Tyr Glu Tyr Ser Phe Ser 405 410
415 gaa agt cat cct gca aaa gga aca ttt ggt gac cat aac ttg act gga
1296 Glu Ser His Pro Ala Lys Gly Thr Phe Gly Asp His Asn Leu Thr
Gly 420 425 430 gca tgt cat ggt gaa gaa ctt gtg aat tta ttc aaa gtc
gag atg atg 1344 Ala Cys His Gly Glu Glu Leu Val Asn Leu Phe Lys
Val Glu Met Met 435 440 445 aag ctg gaa aaa gat aaa ccg aat gtt tta
tta aca aaa gat agg gta 1392 Lys Leu Glu Lys Asp Lys Pro Asn Val
Leu Leu Thr Lys Asp Arg Val 450 455 460 ctt gct atg tgg acg aac ttc
atc aaa aat gga aat cct act cct gaa 1440 Leu Ala Met Trp Thr Asn
Phe Ile Lys Asn Gly Asn Pro Thr Pro Glu 465 470 475 480 gta act gaa
tta ttg cca gtt aaa tgg gaa cct gcc aca aaa gac aag 1488 Val Thr
Glu Leu Leu Pro Val Lys Trp Glu Pro Ala Thr Lys Asp Lys 485 490 495
ttg aat tat ttg aac att gat gcc acc tta act ttg gga aca aat cca
1536 Leu Asn Tyr Leu Asn Ile Asp Ala Thr Leu Thr Leu Gly Thr Asn
Pro 500 505 510 gaa gaa acc cga gtc aaa tty tgg gaa gat gcc aca aaa
act ttg cac 1584 Glu Glu Thr Arg Val Lys Phe Trp Glu Asp Ala Thr
Lys Thr Leu His 515 520 525 agt caa 1590 Ser Gln 530 24 2836 DNA
Ctenocephalides felis CDS (99)..(1889) 24 tagacatgtc gtcttcaaaa
cgtctatttt atcataaaca aaacgagata aataataaca 60 attaagcaac
caaaatgcat taaaaaacac aataaaaa atg tta cct cac agt agt 116 Met Leu
Pro His Ser Ser 1 5 gca tta gtt tta ttt tta ttt ttt tta ttt ttc tta
ttt aca cct atc 164 Ala Leu Val Leu Phe Leu Phe Phe Leu Phe Phe Leu
Phe Thr Pro Ile 10 15 20 ttg tgc ata cta tgg gat aac cta gat cag
cat ttg tgc aga gtt caa 212 Leu Cys Ile Leu Trp Asp Asn Leu Asp Gln
His Leu Cys Arg Val Gln 25 30 35 ttt aac ggg atc acg gaa gga aaa
ccg ttc cga tat aaa gat cat agg 260 Phe Asn Gly Ile Thr Glu Gly Lys
Pro Phe Arg Tyr Lys Asp His Arg 40 45 50 aat gat gta tat tgt tct
tat ttg gga att cct tat gcc gaa ccg cct 308 Asn Asp Val Tyr Cys Ser
Tyr Leu Gly Ile Pro Tyr Ala Glu Pro Pro 55 60 65 70 ttt gga cca tta
cga ttt cag tct cca aaa cca ata tca aat cca aaa 356 Phe Gly Pro Leu
Arg Phe Gln Ser Pro Lys Pro Ile Ser Asn Pro Lys 75 80 85 aca gga
ttc gta cag gct cga act ttg gga gac aaa tgt ttc cag gaa 404 Thr Gly
Phe Val Gln Ala Arg Thr Leu Gly Asp Lys Cys Phe Gln Glu 90 95 100
agt cta ata tat tct tat gca gga agc gaa gat tgc tta tat ctg aat 452
Ser Leu Ile Tyr Ser Tyr Ala Gly Ser Glu Asp Cys Leu Tyr Leu Asn 105
110 115 ata ttc acg cca gag act gtt aat tct gcg aac aat aca aaa tat
cct 500 Ile Phe Thr Pro Glu Thr Val Asn Ser Ala Asn Asn Thr Lys Tyr
Pro 120 125 130 gta atg ttc tgg atc cat gga ggc gca ttc aac caa gga
tca gga tct 548 Val Met Phe Trp Ile His Gly Gly Ala Phe Asn Gln Gly
Ser Gly Ser 135 140 145 150 tat aat ttt ttt gga cct gat tat ttg atc
agg gaa gga att att ttg 596 Tyr Asn Phe Phe Gly Pro Asp Tyr Leu Ile
Arg Glu Gly Ile Ile Leu 155 160 165 gtc act atc aac tat aga tta gga
gtt ttc ggt ttt cta tca gcg ccg 644 Val Thr Ile Asn Tyr Arg Leu Gly
Val Phe Gly Phe Leu Ser Ala Pro 170 175 180 gaa tgg gat atc cat gga
aat atg ggt cta aaa gac cag aga ttg gca 692 Glu Trp Asp Ile His Gly
Asn Met Gly Leu Lys Asp Gln Arg Leu Ala 185 190 195 cta aaa tgg gtt
tac gac aac atc gaa aag ttt ggt gga gac aga gaa 740 Leu Lys Trp Val
Tyr Asp Asn Ile Glu Lys Phe Gly Gly Asp Arg Glu 200 205 210 aaa att
aca att gct gga gaa tct gct gga gca gca agt gtc cat ttt 788 Lys Ile
Thr Ile Ala Gly Glu Ser Ala Gly Ala Ala Ser Val His Phe 215 220 225
230 ctg atg atg gac aac tcg act aga aaa tac tac caa agg gcc att ttg
836 Leu Met Met Asp Asn Ser Thr Arg Lys Tyr Tyr Gln Arg Ala Ile Leu
235 240 245 cag agt ggg aca tta cta aat ccg act gct aat caa att caa
ctt ctg 884 Gln Ser Gly Thr Leu Leu Asn Pro Thr Ala Asn Gln Ile Gln
Leu Leu 250 255 260 cat aga ttt gaa aaa ctc aaa caa gtg cta aac atc
acg caa aaa caa 932 His Arg Phe Glu Lys Leu Lys Gln Val Leu Asn Ile
Thr Gln Lys Gln 265 270 275 gaa ctc cta aac ctg gat aaa aac cta att
tta cga gca gcc tta aac 980 Glu Leu Leu Asn Leu Asp Lys Asn Leu Ile
Leu Arg Ala Ala Leu Asn 280 285 290 aga gtt cct gat agc aac gac cat
gac cga gac aca gta cca gta ttt 1028 Arg Val Pro Asp Ser Asn Asp
His Asp Arg Asp Thr Val Pro Val Phe 295 300 305 310 aat cca gtc tta
gaa tca cca gaa tct cca gat cca ata aca ttt cca 1076 Asn Pro Val
Leu Glu Ser Pro Glu Ser Pro Asp Pro Ile Thr Phe Pro 315 320 325 tct
gcc ttg gaa aga atg aga aat ggt gaa ttt cct gat gtc gat gtc 1124
Ser Ala Leu Glu Arg Met Arg Asn Gly Glu Phe Pro Asp Val Asp Val 330
335 340 atc att ggt ttc aat agt gct gaa ggt tta aga tct atg gca aga
gta 1172 Ile Ile Gly Phe Asn Ser Ala Glu Gly Leu Arg Ser Met Ala
Arg Val 345 350 355 acc aga gga aac atg gaa gtt cac aag act ttg aca
aat ata gaa agg 1220 Thr Arg Gly Asn Met Glu Val His Lys Thr Leu
Thr Asn Ile Glu Arg 360 365 370 gct ata cct aga gat gct aat att tgg
aaa aat cca aat ggt att gag 1268 Ala Ile Pro Arg Asp Ala Asn Ile
Trp Lys Asn Pro Asn Gly Ile Glu 375 380 385 390 gag aaa aaa cta ata
aaa atg ctt aca gag ttt tat gac caa gtg aaa 1316 Glu Lys Lys Leu
Ile Lys Met Leu Thr Glu Phe Tyr Asp Gln Val Lys 395 400 405 gaa caa
aac gat gac att gaa gcc tac gtc caa cta aaa ggc gat gct 1364 Glu
Gln Asn Asp Asp Ile Glu Ala Tyr Val Gln Leu Lys Gly Asp Ala 410 415
420 ggt tac ctc caa gga atc tac cgt acc ttg aaa gcc ata ttt ttc aat
1412 Gly Tyr Leu Gln Gly Ile Tyr Arg Thr Leu Lys Ala Ile Phe Phe
Asn 425 430 435 gaa ttc aga agg aat tcc aat ttg tat ttg tac agg tta
tca gac gat 1460 Glu Phe Arg Arg Asn Ser Asn Leu Tyr Leu Tyr Arg
Leu Ser Asp Asp 440 445 450 acg tat agt gta tat aaa agt tat atc ttg
ccc tat cga tgg ggt tcc 1508 Thr Tyr Ser Val Tyr Lys Ser Tyr Ile
Leu Pro Tyr Arg Trp Gly Ser 455 460 465 470 ttg cca gga gtt agt cat
ggt gat gat tta gga tat ctt ttt gca aac 1556 Leu Pro Gly Val Ser
His Gly Asp Asp Leu Gly Tyr Leu Phe Ala Asn 475 480 485 tcg ttg gat
gtt cct att ttg gga aca acg cac att tct ata ccg caa 1604 Ser Leu
Asp Val Pro Ile Leu Gly Thr Thr His Ile Ser Ile Pro Gln 490 495 500
gat gct atg cag act ctg gaa agg atg gtc agg atc tgg acc aat ttt
1652 Asp Ala Met Gln Thr Leu Glu Arg Met Val Arg Ile Trp Thr Asn
Phe 505 510 515 gta aag aat gga aaa cct aca tca aac act gaa gat gca
tca tgt gat 1700 Val Lys Asn Gly Lys Pro Thr Ser Asn Thr Glu Asp
Ala Ser Cys Asp 520 525 530 aca aaa aga cat tta aac gac att ttt tgg
gaa cca tac aac gac gaa 1748 Thr Lys Arg His Leu Asn Asp Ile Phe
Trp Glu Pro Tyr Asn Asp Glu 535 540 545 550 gaa cca aaa tat ttg gac
atg gga aaa gaa aat ttt gaa atg aaa aat 1796 Glu Pro Lys Tyr Leu
Asp Met Gly Lys Glu Asn Phe Glu Met Lys Asn 555 560 565 att ttg gaa
cta aaa cgc atg atg ctt tgg gat gaa gtt tat aga aat 1844 Ile Leu
Glu Leu Lys Arg Met Met Leu Trp Asp Glu Val Tyr Arg Asn 570 575 580
gcg aat ttg cgg ttt aga gtc tgt aat gaa gaa agt att aga tga 1889
Ala Asn Leu Arg Phe Arg Val Cys Asn Glu Glu Ser Ile Arg 585 590 595
gtttttttaa ttttacatac agccgagagg aaacatgact aaaattggaa agaaaaatca
1949 gaaaaagaaa aatcacatgg accatagtaa ctttattaca tgatttagtt
tcaagtgtat 2009 caagaaaact tattgcatca aagaaaatat tattttgcca
aaattcttgg aaaaacactt 2069 tttatgactg acatggccca taattgaagc
tttttcttct tttaccaaat cgccaaattt 2129 tgtagcgtca gacacattta
tttatgacat ggcaattaat gtgttaaaca ttcaactcta 2189 tattaaaaat
ggtagtattt tctaataaga aggttatata aaaagacttg aaaataataa 2249
gatttgctcg gctatatata aaaacttanc gtctcgttat gctaaacttt tttgatggta
2309 aaaatatgtt gattttccta ataatctaag atattatatt ttagattaaa
ttaaaatatg 2369 atattttcaa ttaattaatt ttagttttaa atgtactata
tttaccagta ctatgaaact 2429 attttaaata tattttttat tacaatattt
atttctcaaa aatgtttagt gtaacaagac 2489 cattaaatta gagttaatgt
tgtaaattaa actatttttt atctatcaca accgcttaat 2549 tggtgcaaag
aaaaatttta ctgtgataat atttgacatt tacacaatat tacgaattgt 2609
aaactcacaa ttatgtgaat attgtttttt gttaaaaaaa catacatgac ttttctatat
2669 cattttatat tacggtgata tggattaatg tcaacatgta aaatacaaat
gcggttgtta 2729 aaaataatct gtattaaaat tgttatataa aatctgaata
aatgtacttt taagtaaaaa 2789 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaa 2836 25 596 PRT Ctenocephalides felis 25 Met Leu
Pro His Ser Ser Ala Leu Val Leu Phe Leu Phe Phe Leu Phe 1 5 10 15
Phe Leu Phe Thr Pro Ile Leu Cys Ile Leu Trp Asp Asn Leu Asp Gln 20
25 30 His Leu Cys Arg Val Gln Phe Asn Gly Ile Thr Glu Gly Lys Pro
Phe 35 40 45 Arg Tyr Lys Asp His Arg Asn Asp Val Tyr Cys Ser Tyr
Leu Gly Ile 50 55 60 Pro Tyr Ala Glu Pro Pro Phe Gly Pro Leu Arg
Phe Gln Ser Pro Lys 65 70 75 80 Pro Ile Ser Asn Pro Lys Thr Gly Phe
Val Gln Ala Arg Thr Leu Gly 85 90 95 Asp Lys Cys Phe Gln Glu Ser
Leu Ile Tyr Ser Tyr Ala Gly Ser Glu 100 105 110 Asp Cys Leu Tyr Leu
Asn Ile Phe Thr Pro Glu Thr Val Asn Ser Ala 115 120 125 Asn Asn Thr
Lys Tyr Pro Val Met Phe Trp Ile His Gly Gly Ala Phe 130 135 140 Asn
Gln Gly Ser Gly Ser Tyr Asn Phe Phe Gly Pro Asp Tyr Leu Ile 145 150
155 160 Arg Glu Gly Ile Ile Leu Val Thr Ile Asn Tyr Arg Leu Gly Val
Phe 165 170 175 Gly Phe Leu Ser Ala Pro Glu Trp Asp Ile His Gly Asn
Met Gly Leu 180 185 190 Lys Asp Gln Arg Leu Ala Leu Lys Trp Val Tyr
Asp Asn Ile Glu Lys 195 200 205 Phe Gly Gly Asp Arg Glu Lys Ile Thr
Ile Ala Gly Glu Ser Ala Gly 210 215 220 Ala Ala Ser Val His Phe Leu
Met Met Asp Asn Ser Thr Arg Lys Tyr 225 230 235 240 Tyr Gln Arg Ala
Ile Leu Gln Ser Gly Thr Leu Leu Asn Pro Thr Ala
245 250 255 Asn Gln Ile Gln Leu Leu His Arg Phe Glu Lys Leu Lys Gln
Val Leu 260 265 270 Asn Ile Thr Gln Lys Gln Glu Leu Leu Asn Leu Asp
Lys Asn Leu Ile 275 280 285 Leu Arg Ala Ala Leu Asn Arg Val Pro Asp
Ser Asn Asp His Asp Arg 290 295 300 Asp Thr Val Pro Val Phe Asn Pro
Val Leu Glu Ser Pro Glu Ser Pro 305 310 315 320 Asp Pro Ile Thr Phe
Pro Ser Ala Leu Glu Arg Met Arg Asn Gly Glu 325 330 335 Phe Pro Asp
Val Asp Val Ile Ile Gly Phe Asn Ser Ala Glu Gly Leu 340 345 350 Arg
Ser Met Ala Arg Val Thr Arg Gly Asn Met Glu Val His Lys Thr 355 360
365 Leu Thr Asn Ile Glu Arg Ala Ile Pro Arg Asp Ala Asn Ile Trp Lys
370 375 380 Asn Pro Asn Gly Ile Glu Glu Lys Lys Leu Ile Lys Met Leu
Thr Glu 385 390 395 400 Phe Tyr Asp Gln Val Lys Glu Gln Asn Asp Asp
Ile Glu Ala Tyr Val 405 410 415 Gln Leu Lys Gly Asp Ala Gly Tyr Leu
Gln Gly Ile Tyr Arg Thr Leu 420 425 430 Lys Ala Ile Phe Phe Asn Glu
Phe Arg Arg Asn Ser Asn Leu Tyr Leu 435 440 445 Tyr Arg Leu Ser Asp
Asp Thr Tyr Ser Val Tyr Lys Ser Tyr Ile Leu 450 455 460 Pro Tyr Arg
Trp Gly Ser Leu Pro Gly Val Ser His Gly Asp Asp Leu 465 470 475 480
Gly Tyr Leu Phe Ala Asn Ser Leu Asp Val Pro Ile Leu Gly Thr Thr 485
490 495 His Ile Ser Ile Pro Gln Asp Ala Met Gln Thr Leu Glu Arg Met
Val 500 505 510 Arg Ile Trp Thr Asn Phe Val Lys Asn Gly Lys Pro Thr
Ser Asn Thr 515 520 525 Glu Asp Ala Ser Cys Asp Thr Lys Arg His Leu
Asn Asp Ile Phe Trp 530 535 540 Glu Pro Tyr Asn Asp Glu Glu Pro Lys
Tyr Leu Asp Met Gly Lys Glu 545 550 555 560 Asn Phe Glu Met Lys Asn
Ile Leu Glu Leu Lys Arg Met Met Leu Trp 565 570 575 Asp Glu Val Tyr
Arg Asn Ala Asn Leu Arg Phe Arg Val Cys Asn Glu 580 585 590 Glu Ser
Ile Arg 595 26 2836 DNA Ctenocephalides felis misc_feature
(559)..(559) n = unknown 26 tttttttttt tttttttttt tttttttttt
tttttttttt tttttttttt ttacttaaaa 60 gtacatttat tcagatttta
tataacaatt ttaatacaga ttatttttaa caaccgcatt 120 tgtattttac
atgttgacat taatccatat caccgtaata taaaatgata tagaaaagtc 180
atgtatgttt ttttaacaaa aaacaatatt cacataattg tgagtttaca attcgtaata
240 ttgtgtaaat gtcaaatatt atcacagtaa aatttttctt tgcaccaatt
aagcggttgt 300 gatagataaa aaatagttta atttacaaca ttaactctaa
tttaatggtc ttgttacact 360 aaacattttt gagaaataaa tattgtaata
aaaaatatat ttaaaatagt ttcatagtac 420 tggtaaatat agtacattta
aaactaaaat taattaattg aaaatatcat attttaattt 480 aatctaaaat
ataatatctt agattattag gaaaatcaac atatttttac catcaaaaaa 540
gtttagcata acgagacgnt aagtttttat atatagccga gcaaatctta ttattttcaa
600 gtctttttat ataaccttct tattagaaaa tactaccatt tttaatatag
agttgaatgt 660 ttaacacatt aattgccatg tcataaataa atgtgtctga
cgctacaaaa tttggcgatt 720 tggtaaaaga agaaaaagct tcaattatgg
gccatgtcag tcataaaaag tgtttttcca 780 agaattttgg caaaataata
ttttctttga tgcaataagt tttcttgata cacttgaaac 840 taaatcatgt
aataaagtta ctatggtcca tgtgattttt ctttttctga tttttctttc 900
caattttagt catgtttcct ctcggctgta tgtaaaatta aaaaaactca tctaatactt
960 tcttcattac agactctaaa ccgcaaattc gcatttctat aaacttcatc
ccaaagcatc 1020 atgcgtttta gttccaaaat atttttcatt tcaaaatttt
cttttcccat gtccaaatat 1080 tttggttctt cgtcgttgta tggttcccaa
aaaatgtcgt ttaaatgtct ttttgtatca 1140 catgatgcat cttcagtgtt
tgatgtaggt tttccattct ttacaaaatt ggtccagatc 1200 ctgaccatcc
tttccagagt ctgcatagca tcttgcggta tagaaatgtg cgttgttccc 1260
aaaataggaa catccaacga gtttgcaaaa agatatccta aatcatcacc atgactaact
1320 cctggcaagg aaccccatcg atagggcaag atataacttt tatatacact
atacgtatcg 1380 tctgataacc tgtacaaata caaattggaa ttccttctga
attcattgaa aaatatggct 1440 ttcaaggtac ggtagattcc ttggaggtaa
ccagcatcgc cttttagttg gacgtaggct 1500 tcaatgtcat cgttttgttc
tttcacttgg tcataaaact ctgtaagcat ttttattagt 1560 tttttctcct
caataccatt tggatttttc caaatattag catctctagg tatagccctt 1620
tctatatttg tcaaagtctt gtgaacttcc atgtttcctc tggttactct tgccatagat
1680 cttaaacctt cagcactatt gaaaccaatg atgacatcga catcaggaaa
ttcaccattt 1740 ctcattcttt ccaaggcaga tggaaatgtt attggatctg
gagattctgg tgattctaag 1800 actggattaa atactggtac tgtgtctcgg
tcatggtcgt tgctatcagg aactctgttt 1860 aaggctgctc gtaaaattag
gtttttatcc aggtttagga gttcttgttt ttgcgtgatg 1920 tttagcactt
gtttgagttt ttcaaatcta tgcagaagtt gaatttgatt agcagtcgga 1980
tttagtaatg tcccactctg caaaatggcc ctttggtagt attttctagt cgagttgtcc
2040 atcatcagaa aatggacact tgctgctcca gcagattctc cagcaattgt
aattttttct 2100 ctgtctccac caaacttttc gatgttgtcg taaacccatt
ttagtgccaa tctctggtct 2160 tttagaccca tatttccatg gatatcccat
tccggcgctg atagaaaacc gaaaactcct 2220 aatctatagt tgatagtgac
caaaataatt ccttccctga tcaaataatc aggtccaaaa 2280 aaattataag
atcctgatcc ttggttgaat gcgcctccat ggatccagaa cattacagga 2340
tattttgtat tgttcgcaga attaacagtc tctggcgtga atatattcag atataagcaa
2400 tcttcgcttc ctgcataaga atatattaga ctttcctgga aacatttgtc
tcccaaagtt 2460 cgagcctgta cgaatcctgt ttttggattt gatattggtt
ttggagactg aaatcgtaat 2520 ggtccaaaag gcggttcggc ataaggaatt
cccaaataag aacaatatac atcattccta 2580 tgatctttat atcggaacgg
ttttccttcc gtgatcccgt taaattgaac tctgcacaaa 2640 tgctgatcta
ggttatccca tagtatgcac aagataggtg taaataagaa aaataaaaaa 2700
aataaaaata aaactaatgc actactgtga ggtaacattt tttattgtgt tttttaatgc
2760 attttggttg cttaattgtt attatttatc tcgttttgtt tatgataaaa
tagacgtttt 2820 gaagacgaca tgtcta 2836 27 1710 DNA Ctenocephalides
felis exon (1)..(1710) 27 tgg gat aac cta gat cag cat ttg tgc aga
gtt caa ttt aac ggg atc 48 Trp Asp Asn Leu Asp Gln His Leu Cys Arg
Val Gln Phe Asn Gly Ile 1 5 10 15 acg gaa gga aaa ccg ttc cga tat
aaa gat cat agg aat gat gta tat 96 Thr Glu Gly Lys Pro Phe Arg Tyr
Lys Asp His Arg Asn Asp Val Tyr 20 25 30 tgt tct tat ttg gga att
cct tat gcc gaa ccg cct ttt gga cca tta 144 Cys Ser Tyr Leu Gly Ile
Pro Tyr Ala Glu Pro Pro Phe Gly Pro Leu 35 40 45 cga ttt cag tct
cca aaa cca ata tca aat cca aaa aca gga ttc gta 192 Arg Phe Gln Ser
Pro Lys Pro Ile Ser Asn Pro Lys Thr Gly Phe Val 50 55 60 cag gct
cga act ttg gga gac aaa tgt ttc cag gaa agt cta ata tat 240 Gln Ala
Arg Thr Leu Gly Asp Lys Cys Phe Gln Glu Ser Leu Ile Tyr 65 70 75 80
tct tat gca gga agc gaa gat tgc tta tat ctg aat ata ttc acg cca 288
Ser Tyr Ala Gly Ser Glu Asp Cys Leu Tyr Leu Asn Ile Phe Thr Pro 85
90 95 gag act gtt aat tct gcg aac aat aca aaa tat cct gta atg ttc
tgg 336 Glu Thr Val Asn Ser Ala Asn Asn Thr Lys Tyr Pro Val Met Phe
Trp 100 105 110 atc cat gga ggc gca ttc aac caa gga tca gga tct tat
aat ttt ttt 384 Ile His Gly Gly Ala Phe Asn Gln Gly Ser Gly Ser Tyr
Asn Phe Phe 115 120 125 gga cct gat tat ttg atc agg gaa gga att att
ttg gtc act atc aac 432 Gly Pro Asp Tyr Leu Ile Arg Glu Gly Ile Ile
Leu Val Thr Ile Asn 130 135 140 tat aga tta gga gtt ttc ggt ttt cta
tca gcg ccg gaa tgg gat atc 480 Tyr Arg Leu Gly Val Phe Gly Phe Leu
Ser Ala Pro Glu Trp Asp Ile 145 150 155 160 cat gga aat atg ggt cta
aaa gac cag aga ttg gca cta aaa tgg gtt 528 His Gly Asn Met Gly Leu
Lys Asp Gln Arg Leu Ala Leu Lys Trp Val 165 170 175 tac gac aac atc
gaa aag ttt ggt gga gac aga gaa aaa att aca att 576 Tyr Asp Asn Ile
Glu Lys Phe Gly Gly Asp Arg Glu Lys Ile Thr Ile 180 185 190 gct gga
gaa tct gct gga gca gca agt gtc cat ttt ctg atg atg gac 624 Ala Gly
Glu Ser Ala Gly Ala Ala Ser Val His Phe Leu Met Met Asp 195 200 205
aac tcg act aga aaa tac tac caa agg gcc att ttg cag agt ggg aca 672
Asn Ser Thr Arg Lys Tyr Tyr Gln Arg Ala Ile Leu Gln Ser Gly Thr 210
215 220 tta cta aat ccg act gct aat caa att caa ctt ctg cat aga ttt
gaa 720 Leu Leu Asn Pro Thr Ala Asn Gln Ile Gln Leu Leu His Arg Phe
Glu 225 230 235 240 aaa ctc aaa caa gtg cta aac atc acg caa aaa caa
gaa ctc cta aac 768 Lys Leu Lys Gln Val Leu Asn Ile Thr Gln Lys Gln
Glu Leu Leu Asn 245 250 255 ctg gat aaa aac cta att tta cga gca gcc
tta aac aga gtt cct gat 816 Leu Asp Lys Asn Leu Ile Leu Arg Ala Ala
Leu Asn Arg Val Pro Asp 260 265 270 agc aac gac cat gac cga gac aca
gta cca gta ttt aat cca gtc tta 864 Ser Asn Asp His Asp Arg Asp Thr
Val Pro Val Phe Asn Pro Val Leu 275 280 285 gaa tca cca gaa tct cca
gat cca ata aca ttt cca tct gcc ttg gaa 912 Glu Ser Pro Glu Ser Pro
Asp Pro Ile Thr Phe Pro Ser Ala Leu Glu 290 295 300 aga atg aga aat
ggt gaa ttt cct gat gtc gat gtc atc att ggt ttc 960 Arg Met Arg Asn
Gly Glu Phe Pro Asp Val Asp Val Ile Ile Gly Phe 305 310 315 320 aat
agt gct gaa ggt tta aga tct atg gca aga gta acc aga gga aac 1008
Asn Ser Ala Glu Gly Leu Arg Ser Met Ala Arg Val Thr Arg Gly Asn 325
330 335 atg gaa gtt cac aag act ttg aca aat ata gaa agg gct ata cct
aga 1056 Met Glu Val His Lys Thr Leu Thr Asn Ile Glu Arg Ala Ile
Pro Arg 340 345 350 gat gct aat att tgg aaa aat cca aat ggt att gag
gag aaa aaa cta 1104 Asp Ala Asn Ile Trp Lys Asn Pro Asn Gly Ile
Glu Glu Lys Lys Leu 355 360 365 ata aaa atg ctt aca gag ttt tat gac
caa gtg aaa gaa caa aac gat 1152 Ile Lys Met Leu Thr Glu Phe Tyr
Asp Gln Val Lys Glu Gln Asn Asp 370 375 380 gac att gaa gcc tac gtc
caa cta aaa ggc gat gct ggt tac ctc caa 1200 Asp Ile Glu Ala Tyr
Val Gln Leu Lys Gly Asp Ala Gly Tyr Leu Gln 385 390 395 400 gga atc
tac cgt acc ttg aaa gcc ata ttt ttc aat gaa ttc aga agg 1248 Gly
Ile Tyr Arg Thr Leu Lys Ala Ile Phe Phe Asn Glu Phe Arg Arg 405 410
415 aat tcc aat ttg tat ttg tac agg tta tca gac gat acg tat agt gta
1296 Asn Ser Asn Leu Tyr Leu Tyr Arg Leu Ser Asp Asp Thr Tyr Ser
Val 420 425 430 tat aaa agt tat atc ttg ccc tat cga tgg ggt tcc ttg
cca gga gtt 1344 Tyr Lys Ser Tyr Ile Leu Pro Tyr Arg Trp Gly Ser
Leu Pro Gly Val 435 440 445 agt cat ggt gat gat tta gga tat ctt ttt
gca aac tcg ttg gat gtt 1392 Ser His Gly Asp Asp Leu Gly Tyr Leu
Phe Ala Asn Ser Leu Asp Val 450 455 460 cct att ttg gga aca acg cac
att tct ata ccg caa gat gct atg cag 1440 Pro Ile Leu Gly Thr Thr
His Ile Ser Ile Pro Gln Asp Ala Met Gln 465 470 475 480 act ctg gaa
agg atg gtc agg atc tgg acc aat ttt gta aag aat gga 1488 Thr Leu
Glu Arg Met Val Arg Ile Trp Thr Asn Phe Val Lys Asn Gly 485 490 495
aaa cct aca tca aac act gaa gat gca tca tgt gat aca aaa aga cat
1536 Lys Pro Thr Ser Asn Thr Glu Asp Ala Ser Cys Asp Thr Lys Arg
His 500 505 510 tta aac gac att ttt tgg gaa cca tac aac gac gaa gaa
cca aaa tat 1584 Leu Asn Asp Ile Phe Trp Glu Pro Tyr Asn Asp Glu
Glu Pro Lys Tyr 515 520 525 ttg gac atg gga aaa gaa aat ttt gaa atg
aaa aat att ttg gaa cta 1632 Leu Asp Met Gly Lys Glu Asn Phe Glu
Met Lys Asn Ile Leu Glu Leu 530 535 540 aaa cgc atg atg ctt tgg gat
gaa gtt tat aga aat gcg aat ttg cgg 1680 Lys Arg Met Met Leu Trp
Asp Glu Val Tyr Arg Asn Ala Asn Leu Arg 545 550 555 560 ttt aga gtc
tgt aat gaa gaa agt att aga 1710 Phe Arg Val Cys Asn Glu Glu Ser
Ile Arg 565 570 28 1788 DNA Ctenocephalides felis exon (1)..(1788)
28 atg tta cct cac agt agt gca tta gtt tta ttt tta ttt ttt tta ttt
48 Met Leu Pro His Ser Ser Ala Leu Val Leu Phe Leu Phe Phe Leu Phe
1 5 10 15 ttc tta ttt aca cct atc ttg tgc ata cta tgg gat aac cta
gat cag 96 Phe Leu Phe Thr Pro Ile Leu Cys Ile Leu Trp Asp Asn Leu
Asp Gln 20 25 30 cat ttg tgc aga gtt caa ttt aac ggg atc acg gaa
gga aaa ccg ttc 144 His Leu Cys Arg Val Gln Phe Asn Gly Ile Thr Glu
Gly Lys Pro Phe 35 40 45 cga tat aaa gat cat agg aat gat gta tat
tgt tct tat ttg gga att 192 Arg Tyr Lys Asp His Arg Asn Asp Val Tyr
Cys Ser Tyr Leu Gly Ile 50 55 60 cct tat gcc gaa ccg cct ttt gga
cca tta cga ttt cag tct cca aaa 240 Pro Tyr Ala Glu Pro Pro Phe Gly
Pro Leu Arg Phe Gln Ser Pro Lys 65 70 75 80 cca ata tca aat cca aaa
aca gga ttc gta cag gct cga act ttg gga 288 Pro Ile Ser Asn Pro Lys
Thr Gly Phe Val Gln Ala Arg Thr Leu Gly 85 90 95 gac aaa tgt ttc
cag gaa agt cta ata tat tct tat gca gga agc gaa 336 Asp Lys Cys Phe
Gln Glu Ser Leu Ile Tyr Ser Tyr Ala Gly Ser Glu 100 105 110 gat tgc
tta tat ctg aat ata ttc acg cca gag act gtt aat tct gcg 384 Asp Cys
Leu Tyr Leu Asn Ile Phe Thr Pro Glu Thr Val Asn Ser Ala 115 120 125
aac aat aca aaa tat cct gta atg ttc tgg atc cat gga ggc gca ttc 432
Asn Asn Thr Lys Tyr Pro Val Met Phe Trp Ile His Gly Gly Ala Phe 130
135 140 aac caa gga tca gga tct tat aat ttt ttt gga cct gat tat ttg
atc 480 Asn Gln Gly Ser Gly Ser Tyr Asn Phe Phe Gly Pro Asp Tyr Leu
Ile 145 150 155 160 agg gaa gga att att ttg gtc act atc aac tat aga
tta gga gtt ttc 528 Arg Glu Gly Ile Ile Leu Val Thr Ile Asn Tyr Arg
Leu Gly Val Phe 165 170 175 ggt ttt cta tca gcg ccg gaa tgg gat atc
cat gga aat atg ggt cta 576 Gly Phe Leu Ser Ala Pro Glu Trp Asp Ile
His Gly Asn Met Gly Leu 180 185 190 aaa gac cag aga ttg gca cta aaa
tgg gtt tac gac aac atc gaa aag 624 Lys Asp Gln Arg Leu Ala Leu Lys
Trp Val Tyr Asp Asn Ile Glu Lys 195 200 205 ttt ggt gga gac aga gaa
aaa att aca att gct gga gaa tct gct gga 672 Phe Gly Gly Asp Arg Glu
Lys Ile Thr Ile Ala Gly Glu Ser Ala Gly 210 215 220 gca gca agt gtc
cat ttt ctg atg atg gac aac tcg act aga aaa tac 720 Ala Ala Ser Val
His Phe Leu Met Met Asp Asn Ser Thr Arg Lys Tyr 225 230 235 240 tac
caa agg gcc att ttg cag agt ggg aca tta cta aat ccg act gct 768 Tyr
Gln Arg Ala Ile Leu Gln Ser Gly Thr Leu Leu Asn Pro Thr Ala 245 250
255 aat caa att caa ctt ctg cat aga ttt gaa aaa ctc aaa caa gtg cta
816 Asn Gln Ile Gln Leu Leu His Arg Phe Glu Lys Leu Lys Gln Val Leu
260 265 270 aac atc acg caa aaa caa gaa ctc cta aac ctg gat aaa aac
cta att 864 Asn Ile Thr Gln Lys Gln Glu Leu Leu Asn Leu Asp Lys Asn
Leu Ile 275 280 285 tta cga gca gcc tta aac aga gtt cct gat agc aac
gac cat gac cga 912 Leu Arg Ala Ala Leu Asn Arg Val Pro Asp Ser Asn
Asp His Asp Arg 290 295 300 gac aca gta cca gta ttt aat cca gtc tta
gaa tca cca gaa tct cca 960 Asp Thr Val Pro Val Phe Asn Pro Val Leu
Glu Ser Pro Glu Ser Pro 305 310 315 320 gat cca ata aca ttt cca tct
gcc ttg gaa aga atg aga aat ggt gaa 1008 Asp Pro Ile Thr Phe Pro
Ser Ala Leu Glu Arg Met Arg Asn Gly Glu 325 330 335 ttt cct gat gtc
gat gtc atc att ggt ttc aat agt gct gaa ggt tta 1056 Phe Pro Asp
Val Asp Val Ile Ile Gly Phe Asn Ser Ala Glu Gly Leu 340 345 350 aga
tct atg gca aga gta acc aga gga aac atg gaa gtt cac aag act 1104
Arg Ser Met Ala Arg Val Thr Arg Gly Asn Met Glu Val His Lys Thr 355
360 365 ttg aca aat ata gaa agg gct ata cct aga gat gct aat att tgg
aaa 1152 Leu Thr Asn Ile Glu Arg Ala Ile Pro Arg Asp Ala Asn Ile
Trp Lys 370 375 380 aat cca aat ggt att gag gag aaa aaa cta ata aaa
atg ctt aca gag 1200 Asn Pro Asn Gly Ile Glu Glu Lys Lys Leu Ile
Lys Met Leu Thr Glu 385 390 395 400 ttt tat gac caa gtg aaa gaa caa
aac gat gac att gaa gcc tac gtc 1248 Phe Tyr Asp Gln Val Lys Glu
Gln Asn Asp Asp Ile Glu Ala Tyr Val 405 410 415 caa cta aaa ggc gat
gct ggt tac ctc caa gga atc tac cgt acc ttg 1296 Gln Leu Lys Gly
Asp Ala Gly Tyr Leu Gln Gly Ile
Tyr Arg Thr Leu 420 425 430 aaa gcc ata ttt ttc aat gaa ttc aga agg
aat tcc aat ttg tat ttg 1344 Lys Ala Ile Phe Phe Asn Glu Phe Arg
Arg Asn Ser Asn Leu Tyr Leu 435 440 445 tac agg tta tca gac gat acg
tat agt gta tat aaa agt tat atc ttg 1392 Tyr Arg Leu Ser Asp Asp
Thr Tyr Ser Val Tyr Lys Ser Tyr Ile Leu 450 455 460 ccc tat cga tgg
ggt tcc ttg cca gga gtt agt cat ggt gat gat tta 1440 Pro Tyr Arg
Trp Gly Ser Leu Pro Gly Val Ser His Gly Asp Asp Leu 465 470 475 480
gga tat ctt ttt gca aac tcg ttg gat gtt cct att ttg gga aca acg
1488 Gly Tyr Leu Phe Ala Asn Ser Leu Asp Val Pro Ile Leu Gly Thr
Thr 485 490 495 cac att tct ata ccg caa gat gct atg cag act ctg gaa
agg atg gtc 1536 His Ile Ser Ile Pro Gln Asp Ala Met Gln Thr Leu
Glu Arg Met Val 500 505 510 agg atc tgg acc aat ttt gta aag aat gga
aaa cct aca tca aac act 1584 Arg Ile Trp Thr Asn Phe Val Lys Asn
Gly Lys Pro Thr Ser Asn Thr 515 520 525 gaa gat gca tca tgt gat aca
aaa aga cat tta aac gac att ttt tgg 1632 Glu Asp Ala Ser Cys Asp
Thr Lys Arg His Leu Asn Asp Ile Phe Trp 530 535 540 gaa cca tac aac
gac gaa gaa cca aaa tat ttg gac atg gga aaa gaa 1680 Glu Pro Tyr
Asn Asp Glu Glu Pro Lys Tyr Leu Asp Met Gly Lys Glu 545 550 555 560
aat ttt gaa atg aaa aat att ttg gaa cta aaa cgc atg atg ctt tgg
1728 Asn Phe Glu Met Lys Asn Ile Leu Glu Leu Lys Arg Met Met Leu
Trp 565 570 575 gat gaa gtt tat aga aat gcg aat ttg cgg ttt aga gtc
tgt aat gaa 1776 Asp Glu Val Tyr Arg Asn Ala Asn Leu Arg Phe Arg
Val Cys Asn Glu 580 585 590 gaa agt att aga 1788 Glu Ser Ile Arg
595 29 1788 DNA Ctenocephalides felis 29 tctaatactt tcttcattac
agactctaaa ccgcaaattc gcatttctat aaacttcatc 60 ccaaagcatc
atgcgtttta gttccaaaat atttttcatt tcaaaatttt cttttcccat 120
gtccaaatat tttggttctt cgtcgttgta tggttcccaa aaaatgtcgt ttaaatgtct
180 ttttgtatca catgatgcat cttcagtgtt tgatgtaggt tttccattct
ttacaaaatt 240 ggtccagatc ctgaccatcc tttccagagt ctgcatagca
tcttgcggta tagaaatgtg 300 cgttgttccc aaaataggaa catccaacga
gtttgcaaaa agatatccta aatcatcacc 360 atgactaact cctggcaagg
aaccccatcg atagggcaag atataacttt tatatacact 420 atacgtatcg
tctgataacc tgtacaaata caaattggaa ttccttctga attcattgaa 480
aaatatggct ttcaaggtac ggtagattcc ttggaggtaa ccagcatcgc cttttagttg
540 gacgtaggct tcaatgtcat cgttttgttc tttcacttgg tcataaaact
ctgtaagcat 600 ttttattagt tttttctcct caataccatt tggatttttc
caaatattag catctctagg 660 tatagccctt tctatatttg tcaaagtctt
gtgaacttcc atgtttcctc tggttactct 720 tgccatagat cttaaacctt
cagcactatt gaaaccaatg atgacatcga catcaggaaa 780 ttcaccattt
ctcattcttt ccaaggcaga tggaaatgtt attggatctg gagattctgg 840
tgattctaag actggattaa atactggtac tgtgtctcgg tcatggtcgt tgctatcagg
900 aactctgttt aaggctgctc gtaaaattag gtttttatcc aggtttagga
gttcttgttt 960 ttgcgtgatg tttagcactt gtttgagttt ttcaaatcta
tgcagaagtt gaatttgatt 1020 agcagtcgga tttagtaatg tcccactctg
caaaatggcc ctttggtagt attttctagt 1080 cgagttgtcc atcatcagaa
aatggacact tgctgctcca gcagattctc cagcaattgt 1140 aattttttct
ctgtctccac caaacttttc gatgttgtcg taaacccatt ttagtgccaa 1200
tctctggtct tttagaccca tatttccatg gatatcccat tccggcgctg atagaaaacc
1260 gaaaactcct aatctatagt tgatagtgac caaaataatt ccttccctga
tcaaataatc 1320 aggtccaaaa aaattataag atcctgatcc ttggttgaat
gcgcctccat ggatccagaa 1380 cattacagga tattttgtat tgttcgcaga
attaacagtc tctggcgtga atatattcag 1440 atataagcaa tcttcgcttc
ctgcataaga atatattaga ctttcctgga aacatttgtc 1500 tcccaaagtt
cgagcctgta cgaatcctgt ttttggattt gatattggtt ttggagactg 1560
aaatcgtaat ggtccaaaag gcggttcggc ataaggaatt cccaaataag aacaatatac
1620 atcattccta tgatctttat atcggaacgg ttttccttcc gtgatcccgt
taaattgaac 1680 tctgcacaaa tgctgatcta ggttatccca tagtatgcac
aagataggtg taaataagaa 1740 aaataaaaaa aataaaaata aaactaatgc
actactgtga ggtaacat 1788 30 2801 DNA Ctenocephalides felis CDS
(99)..(1886) 30 gacatgtcgt cttcaaaacg tctattttat cataaacaaa
acgagataaa taataacaat 60 taagcatcca aaatgcatta aaaaaaacat cataaaaa
atg tta cct cac agt gca 116 Met Leu Pro His Ser Ala 1 5 tta gtt tta
ttt tta ttt ttt tta ttt ttc tta ttt aca cct gtc ttg 164 Leu Val Leu
Phe Leu Phe Phe Leu Phe Phe Leu Phe Thr Pro Val Leu 10 15 20 tgc
ata cta tgg gat aac cta gat cag cat ttg tgc aga gtt caa ttt 212 Cys
Ile Leu Trp Asp Asn Leu Asp Gln His Leu Cys Arg Val Gln Phe 25 30
35 aac ggg atc acg gaa gga aaa ccg ttc cga tat aaa gat cat aaa aat
260 Asn Gly Ile Thr Glu Gly Lys Pro Phe Arg Tyr Lys Asp His Lys Asn
40 45 50 gat gta tat tgt tcc tat ttg gga att cct tat gca gaa ccg
cct att 308 Asp Val Tyr Cys Ser Tyr Leu Gly Ile Pro Tyr Ala Glu Pro
Pro Ile 55 60 65 70 gga cca ttg cga ttt cag tct cca aaa cca ata tca
aat cca aaa aca 356 Gly Pro Leu Arg Phe Gln Ser Pro Lys Pro Ile Ser
Asn Pro Lys Thr 75 80 85 gga ttc gtt cag gct cgg tct tta gga gac
aaa tgt ttc cag gaa agt 404 Gly Phe Val Gln Ala Arg Ser Leu Gly Asp
Lys Cys Phe Gln Glu Ser 90 95 100 cta ata tat tct tat gca gga agc
gaa gat tgc tta tat ctg aat ata 452 Leu Ile Tyr Ser Tyr Ala Gly Ser
Glu Asp Cys Leu Tyr Leu Asn Ile 105 110 115 ttc acg cca gag act gtt
aat tct gcg aac aat aca aaa tat cct gta 500 Phe Thr Pro Glu Thr Val
Asn Ser Ala Asn Asn Thr Lys Tyr Pro Val 120 125 130 atg ttc tgg atc
cat gga ggc gca ttc aac caa gga tca gga tct tat 548 Met Phe Trp Ile
His Gly Gly Ala Phe Asn Gln Gly Ser Gly Ser Tyr 135 140 145 150 aat
ttt ttt gga cct gat tat ttg atc agg gaa gga att att ttg gtc 596 Asn
Phe Phe Gly Pro Asp Tyr Leu Ile Arg Glu Gly Ile Ile Leu Val 155 160
165 act atc aac tat aga tta gga gtt ttc ggt ttt cta tca gcg ccg gaa
644 Thr Ile Asn Tyr Arg Leu Gly Val Phe Gly Phe Leu Ser Ala Pro Glu
170 175 180 tgg gat atc cat gga aat atg ggt cta aaa gac cag aga ttg
gca cta 692 Trp Asp Ile His Gly Asn Met Gly Leu Lys Asp Gln Arg Leu
Ala Leu 185 190 195 aaa tgg gtt tat gac aac atc gaa aaa ttt ggt gga
gac aga gat aaa 740 Lys Trp Val Tyr Asp Asn Ile Glu Lys Phe Gly Gly
Asp Arg Asp Lys 200 205 210 atc act ata gct gga gaa tct gct gga gca
gca agt gtt cat ttt ctg 788 Ile Thr Ile Ala Gly Glu Ser Ala Gly Ala
Ala Ser Val His Phe Leu 215 220 225 230 atg atg gac aat tct act aga
aaa tac tac caa agg gca att ttg cag 836 Met Met Asp Asn Ser Thr Arg
Lys Tyr Tyr Gln Arg Ala Ile Leu Gln 235 240 245 agt ggg aca tta ctc
aat ccg act gct aat caa att caa cct ctg cat 884 Ser Gly Thr Leu Leu
Asn Pro Thr Ala Asn Gln Ile Gln Pro Leu His 250 255 260 aga ttt gaa
aaa cta aaa caa gtg ctg aac atc acg caa aaa caa gaa 932 Arg Phe Glu
Lys Leu Lys Gln Val Leu Asn Ile Thr Gln Lys Gln Glu 265 270 275 ctc
cta aat ctg gac aaa aat caa att ttg cga gca gcc tta aac aga 980 Leu
Leu Asn Leu Asp Lys Asn Gln Ile Leu Arg Ala Ala Leu Asn Arg 280 285
290 gtc cca gat aac aac gac cac gaa agg gac aca gta cca gta ttt aat
1028 Val Pro Asp Asn Asn Asp His Glu Arg Asp Thr Val Pro Val Phe
Asn 295 300 305 310 cca gtc cta gaa tca cca gaa tct cca gac cca ata
aca ttt cca tct 1076 Pro Val Leu Glu Ser Pro Glu Ser Pro Asp Pro
Ile Thr Phe Pro Ser 315 320 325 gct tta gaa aga atg aga aat ggt gaa
ttt cct gac gtt gat gtc atc 1124 Ala Leu Glu Arg Met Arg Asn Gly
Glu Phe Pro Asp Val Asp Val Ile 330 335 340 att gga ttc aat agt gct
gaa ggt tta aga tct atg cca aga gta acc 1172 Ile Gly Phe Asn Ser
Ala Glu Gly Leu Arg Ser Met Pro Arg Val Thr 345 350 355 aga gga aac
atg gaa gtt tac aag act ttg aca aat ata gag aga gct 1220 Arg Gly
Asn Met Glu Val Tyr Lys Thr Leu Thr Asn Ile Glu Arg Ala 360 365 370
ata cct aga gat gct aat att tgg aaa aat cct aat ggc att gag gag
1268 Ile Pro Arg Asp Ala Asn Ile Trp Lys Asn Pro Asn Gly Ile Glu
Glu 375 380 385 390 aaa aaa ctt ata aaa atg ctt aca gag ttt tat gac
caa gtt aaa gaa 1316 Lys Lys Leu Ile Lys Met Leu Thr Glu Phe Tyr
Asp Gln Val Lys Glu 395 400 405 caa aac gat gac atc gaa gcc tat gtc
caa cta aaa ggc gat gct ggt 1364 Gln Asn Asp Asp Ile Glu Ala Tyr
Val Gln Leu Lys Gly Asp Ala Gly 410 415 420 tat ctc caa gga att tac
cgt acc ttg aaa gcc ata ttt ttc aat gaa 1412 Tyr Leu Gln Gly Ile
Tyr Arg Thr Leu Lys Ala Ile Phe Phe Asn Glu 425 430 435 atc aaa aga
aat tcc aac ttg tat ttg tat agg tta tca gat gat acg 1460 Ile Lys
Arg Asn Ser Asn Leu Tyr Leu Tyr Arg Leu Ser Asp Asp Thr 440 445 450
tat agt gta tat aaa agt tat atc ttg ccc tat cga tgg ggt tcc ttg
1508 Tyr Ser Val Tyr Lys Ser Tyr Ile Leu Pro Tyr Arg Trp Gly Ser
Leu 455 460 465 470 cca gga gtt agt cat ggt gat gat tta gga tat ctt
ttt gca aac tct 1556 Pro Gly Val Ser His Gly Asp Asp Leu Gly Tyr
Leu Phe Ala Asn Ser 475 480 485 ttg gat gtt cct att ttg gga aca acg
cac att tct ata ccg caa gat 1604 Leu Asp Val Pro Ile Leu Gly Thr
Thr His Ile Ser Ile Pro Gln Asp 490 495 500 gct atg cag act ctg gaa
agg atg gtc agg atc tgg acc aat ttt gta 1652 Ala Met Gln Thr Leu
Glu Arg Met Val Arg Ile Trp Thr Asn Phe Val 505 510 515 aag aat gga
aaa cct aca tca aac act gaa gat gca tca tgt gat aca 1700 Lys Asn
Gly Lys Pro Thr Ser Asn Thr Glu Asp Ala Ser Cys Asp Thr 520 525 530
aaa aga cat tta aac gac att ttt tgg gaa cca tac aac gac gaa gaa
1748 Lys Arg His Leu Asn Asp Ile Phe Trp Glu Pro Tyr Asn Asp Glu
Glu 535 540 545 550 cca aaa tat ttg gac atg gga aaa gaa cat ttt gaa
atg aaa aat att 1796 Pro Lys Tyr Leu Asp Met Gly Lys Glu His Phe
Glu Met Lys Asn Ile 555 560 565 ttg gaa cta aaa cgc atg atg ctt tgg
gat gaa gtt tat aga aat gcg 1844 Leu Glu Leu Lys Arg Met Met Leu
Trp Asp Glu Val Tyr Arg Asn Ala 570 575 580 aat ttg cgg ttt aga gtc
tgt aat gaa gaa agt att aga tga 1886 Asn Leu Arg Phe Arg Val Cys
Asn Glu Glu Ser Ile Arg 585 590 595 gtttttttaa ttttacatac
agccgagagg aaacatgact aaaattggaa agaaaaatca 1946 gaaaaagaaa
aatcacatgg accatagtaa ctttattaca tgatttagtt tcaagtgtat 2006
caagaaaact tattgcatca aagaaaatat tattttgcca aaattcttgg aaaaacactt
2066 tttatgactg acatggccca taattgaagc tttttcttct tttaccaaat
cgccaaattt 2126 tgtagcgtca gacacattta tttatgacat ggcaattaat
gtgttaaaca ttcaactcta 2186 tattaaaaat ggtagtattt tctaataaga
aggttatata aaaagacttg aaaataataa 2246 gatttgctcg gctatatata
aaaacttanc gtctcgttat gctaaacttt tttgatggta 2306 aaaatatgtt
gattttccta ataatctaag atattatatt ttagattaaa ttaaaatatg 2366
atattttcaa ttaattaatt ttagttttaa atgtactata tttaccagta ctatgaaact
2426 attttaaata tattttttat tacaatattt atttctcaaa aatgtttagt
gtaacaagac 2486 cattaaatta gagttaatgt tgtaaattaa actatttttt
atctatcaca accgcttaat 2546 tggtgcaaag aaaaatttta ctgtgataat
atttgacatt tacacaatat tacgaattgt 2606 aaactcacaa ttatgtgaat
attgtttttt gttaaaaaaa catacatgac ttttctatat 2666 cattttatat
tacggtgata tggattaatg tcaacatgta aaatacaaat gcggttgtta 2726
aaaataatct gtattaaaat tgttatataa aatctgaata aatgtacttt taagtaaaaa
2786 aaaaaaaaaa aaaaa 2801 31 595 PRT Ctenocephalides felis 31 Met
Leu Pro His Ser Ala Leu Val Leu Phe Leu Phe Phe Leu Phe Phe 1 5 10
15 Leu Phe Thr Pro Val Leu Cys Ile Leu Trp Asp Asn Leu Asp Gln His
20 25 30 Leu Cys Arg Val Gln Phe Asn Gly Ile Thr Glu Gly Lys Pro
Phe Arg 35 40 45 Tyr Lys Asp His Lys Asn Asp Val Tyr Cys Ser Tyr
Leu Gly Ile Pro 50 55 60 Tyr Ala Glu Pro Pro Ile Gly Pro Leu Arg
Phe Gln Ser Pro Lys Pro 65 70 75 80 Ile Ser Asn Pro Lys Thr Gly Phe
Val Gln Ala Arg Ser Leu Gly Asp 85 90 95 Lys Cys Phe Gln Glu Ser
Leu Ile Tyr Ser Tyr Ala Gly Ser Glu Asp 100 105 110 Cys Leu Tyr Leu
Asn Ile Phe Thr Pro Glu Thr Val Asn Ser Ala Asn 115 120 125 Asn Thr
Lys Tyr Pro Val Met Phe Trp Ile His Gly Gly Ala Phe Asn 130 135 140
Gln Gly Ser Gly Ser Tyr Asn Phe Phe Gly Pro Asp Tyr Leu Ile Arg 145
150 155 160 Glu Gly Ile Ile Leu Val Thr Ile Asn Tyr Arg Leu Gly Val
Phe Gly 165 170 175 Phe Leu Ser Ala Pro Glu Trp Asp Ile His Gly Asn
Met Gly Leu Lys 180 185 190 Asp Gln Arg Leu Ala Leu Lys Trp Val Tyr
Asp Asn Ile Glu Lys Phe 195 200 205 Gly Gly Asp Arg Asp Lys Ile Thr
Ile Ala Gly Glu Ser Ala Gly Ala 210 215 220 Ala Ser Val His Phe Leu
Met Met Asp Asn Ser Thr Arg Lys Tyr Tyr 225 230 235 240 Gln Arg Ala
Ile Leu Gln Ser Gly Thr Leu Leu Asn Pro Thr Ala Asn 245 250 255 Gln
Ile Gln Pro Leu His Arg Phe Glu Lys Leu Lys Gln Val Leu Asn 260 265
270 Ile Thr Gln Lys Gln Glu Leu Leu Asn Leu Asp Lys Asn Gln Ile Leu
275 280 285 Arg Ala Ala Leu Asn Arg Val Pro Asp Asn Asn Asp His Glu
Arg Asp 290 295 300 Thr Val Pro Val Phe Asn Pro Val Leu Glu Ser Pro
Glu Ser Pro Asp 305 310 315 320 Pro Ile Thr Phe Pro Ser Ala Leu Glu
Arg Met Arg Asn Gly Glu Phe 325 330 335 Pro Asp Val Asp Val Ile Ile
Gly Phe Asn Ser Ala Glu Gly Leu Arg 340 345 350 Ser Met Pro Arg Val
Thr Arg Gly Asn Met Glu Val Tyr Lys Thr Leu 355 360 365 Thr Asn Ile
Glu Arg Ala Ile Pro Arg Asp Ala Asn Ile Trp Lys Asn 370 375 380 Pro
Asn Gly Ile Glu Glu Lys Lys Leu Ile Lys Met Leu Thr Glu Phe 385 390
395 400 Tyr Asp Gln Val Lys Glu Gln Asn Asp Asp Ile Glu Ala Tyr Val
Gln 405 410 415 Leu Lys Gly Asp Ala Gly Tyr Leu Gln Gly Ile Tyr Arg
Thr Leu Lys 420 425 430 Ala Ile Phe Phe Asn Glu Ile Lys Arg Asn Ser
Asn Leu Tyr Leu Tyr 435 440 445 Arg Leu Ser Asp Asp Thr Tyr Ser Val
Tyr Lys Ser Tyr Ile Leu Pro 450 455 460 Tyr Arg Trp Gly Ser Leu Pro
Gly Val Ser His Gly Asp Asp Leu Gly 465 470 475 480 Tyr Leu Phe Ala
Asn Ser Leu Asp Val Pro Ile Leu Gly Thr Thr His 485 490 495 Ile Ser
Ile Pro Gln Asp Ala Met Gln Thr Leu Glu Arg Met Val Arg 500 505 510
Ile Trp Thr Asn Phe Val Lys Asn Gly Lys Pro Thr Ser Asn Thr Glu 515
520 525 Asp Ala Ser Cys Asp Thr Lys Arg His Leu Asn Asp Ile Phe Trp
Glu 530 535 540 Pro Tyr Asn Asp Glu Glu Pro Lys Tyr Leu Asp Met Gly
Lys Glu His 545 550 555 560 Phe Glu Met Lys Asn Ile Leu Glu Leu Lys
Arg Met Met Leu Trp Asp 565 570 575 Glu Val Tyr Arg Asn Ala Asn Leu
Arg Phe Arg Val Cys Asn Glu Glu 580 585 590 Ser Ile Arg 595 32 2801
DNA Ctenocephalides felis misc_feature (527)..(527) n = unknown at
position 527 32 tttttttttt tttttttttt acttaaaagt acatttattc
agattttata taacaatttt 60 aatacagatt atttttaaca accgcatttg
tattttacat gttgacatta atccatatca 120 ccgtaatata aaatgatata
gaaaagtcat gtatgttttt ttaacaaaaa acaatattca 180 cataattgtg
agtttacaat tcgtaatatt gtgtaaatgt caaatattat cacagtaaaa 240
tttttctttg caccaattaa gcggttgtga tagataaaaa atagtttaat ttacaacatt
300 aactctaatt taatggtctt gttacactaa acatttttga gaaataaata
ttgtaataaa 360 aaatatattt aaaatagttt catagtactg gtaaatatag
tacatttaaa actaaaatta 420 attaattgaa aatatcatat tttaatttaa
tctaaaatat aatatcttag attattagga 480 aaatcaacat atttttacca
tcaaaaaagt ttagcataac gagacgntaa gtttttatat 540 atagccgagc
aaatcttatt attttcaagt ctttttatat aaccttctta ttagaaaata 600
ctaccatttt taatatagag ttgaatgttt aacacattaa ttgccatgtc
ataaataaat
660 gtgtctgacg ctacaaaatt tggcgatttg gtaaaagaag aaaaagcttc
aattatgggc 720 catgtcagtc ataaaaagtg tttttccaag aattttggca
aaataatatt ttctttgatg 780 caataagttt tcttgataca cttgaaacta
aatcatgtaa taaagttact atggtccatg 840 tgatttttct ttttctgatt
tttctttcca attttagtca tgtttcctct cggctgtatg 900 taaaattaaa
aaaactcatc taatactttc ttcattacag actctaaacc gcaaattcgc 960
atttctataa acttcatccc aaagcatcat gcgttttagt tccaaaatat ttttcatttc
1020 aaaatgttct tttcccatgt ccaaatattt tggttcttcg tcgttgtatg
gttcccaaaa 1080 aatgtcgttt aaatgtcttt ttgtatcaca tgatgcatct
tcagtgtttg atgtaggttt 1140 tccattcttt acaaaattgg tccagatcct
gaccatcctt tccagagtct gcatagcatc 1200 ttgcggtata gaaatgtgcg
ttgttcccaa aataggaaca tccaaagagt ttgcaaaaag 1260 atatcctaaa
tcatcaccat gactaactcc tggcaaggaa ccccatcgat agggcaagat 1320
ataactttta tatacactat acgtatcatc tgataaccta tacaaataca agttggaatt
1380 tcttttgatt tcattgaaaa atatggcttt caaggtacgg taaattcctt
ggagataacc 1440 agcatcgcct tttagttgga cataggcttc gatgtcatcg
ttttgttctt taacttggtc 1500 ataaaactct gtaagcattt ttataagttt
tttctcctca atgccattag gatttttcca 1560 aatattagca tctctaggta
tagctctctc tatatttgtc aaagtcttgt aaacttccat 1620 gtttcctctg
gttactcttg gcatagatct taaaccttca gcactattga atccaatgat 1680
gacatcaacg tcaggaaatt caccatttct cattctttct aaagcagatg gaaatgttat
1740 tgggtctgga gattctggtg attctaggac tggattaaat actggtactg
tgtccctttc 1800 gtggtcgttg ttatctggga ctctgtttaa ggctgctcgc
aaaatttgat ttttgtccag 1860 atttaggagt tcttgttttt gcgtgatgtt
cagcacttgt tttagttttt caaatctatg 1920 cagaggttga atttgattag
cagtcggatt gagtaatgtc ccactctgca aaattgccct 1980 ttggtagtat
tttctagtag aattgtccat catcagaaaa tgaacacttg ctgctccagc 2040
agattctcca gctatagtga ttttatctct gtctccacca aatttttcga tgttgtcata
2100 aacccatttt agtgccaatc tctggtcttt tagacccata tttccatgga
tatcccattc 2160 cggcgctgat agaaaaccga aaactcctaa tctatagttg
atagtgacca aaataattcc 2220 ttccctgatc aaataatcag gtccaaaaaa
attataagat cctgatcctt ggttgaatgc 2280 gcctccatgg atccagaaca
ttacaggata ttttgtattg ttcgcagaat taacagtctc 2340 tggcgtgaat
atattcagat ataagcaatc ttcgcttcct gcataagaat atattagact 2400
ttcctggaaa catttgtctc ctaaagaccg agcctgaacg aatcctgttt ttggatttga
2460 tattggtttt ggagactgaa atcgcaatgg tccaataggc ggttctgcat
aaggaattcc 2520 caaataggaa caatatacat catttttatg atctttatat
cggaacggtt ttccttccgt 2580 gatcccgtta aattgaactc tgcacaaatg
ctgatctagg ttatcccata gtatgcacaa 2640 gacaggtgta aataagaaaa
ataaaaaaaa taaaaataaa actaatgcac tgtgaggtaa 2700 cattttttat
gatgtttttt ttaatgcatt ttggatgctt aattgttatt atttatctcg 2760
ttttgtttat gataaaatag acgttttgaa gacgacatgt c 2801 33 1710 DNA
Ctenocephalides felis exon (1)..(1710) 33 tgg gat aac cta gat cag
cat ttg tgc aga gtt caa ttt aac ggg atc 48 Trp Asp Asn Leu Asp Gln
His Leu Cys Arg Val Gln Phe Asn Gly Ile 1 5 10 15 acg gaa gga aaa
ccg ttc cga tat aaa gat cat aaa aat gat gta tat 96 Thr Glu Gly Lys
Pro Phe Arg Tyr Lys Asp His Lys Asn Asp Val Tyr 20 25 30 tgt tcc
tat ttg gga att cct tat gca gaa ccg cct att gga cca ttg 144 Cys Ser
Tyr Leu Gly Ile Pro Tyr Ala Glu Pro Pro Ile Gly Pro Leu 35 40 45
cga ttt cag tct cca aaa cca ata tca aat cca aaa aca gga ttc gtt 192
Arg Phe Gln Ser Pro Lys Pro Ile Ser Asn Pro Lys Thr Gly Phe Val 50
55 60 cag gct cgg tct tta gga gac aaa tgt ttc cag gaa agt cta ata
tat 240 Gln Ala Arg Ser Leu Gly Asp Lys Cys Phe Gln Glu Ser Leu Ile
Tyr 65 70 75 80 tct tat gca gga agc gaa gat tgc tta tat ctg aat ata
ttc acg cca 288 Ser Tyr Ala Gly Ser Glu Asp Cys Leu Tyr Leu Asn Ile
Phe Thr Pro 85 90 95 gag act gtt aat tct gcg aac aat aca aaa tat
cct gta atg ttc tgg 336 Glu Thr Val Asn Ser Ala Asn Asn Thr Lys Tyr
Pro Val Met Phe Trp 100 105 110 atc cat gga ggc gca ttc aac caa gga
tca gga tct tat aat ttt ttt 384 Ile His Gly Gly Ala Phe Asn Gln Gly
Ser Gly Ser Tyr Asn Phe Phe 115 120 125 gga cct gat tat ttg atc agg
gaa gga att att ttg gtc act atc aac 432 Gly Pro Asp Tyr Leu Ile Arg
Glu Gly Ile Ile Leu Val Thr Ile Asn 130 135 140 tat aga tta gga gtt
ttc ggt ttt cta tca gcg ccg gaa tgg gat atc 480 Tyr Arg Leu Gly Val
Phe Gly Phe Leu Ser Ala Pro Glu Trp Asp Ile 145 150 155 160 cat gga
aat atg ggt cta aaa gac cag aga ttg gca cta aaa tgg gtt 528 His Gly
Asn Met Gly Leu Lys Asp Gln Arg Leu Ala Leu Lys Trp Val 165 170 175
tat gac aac atc gaa aaa ttt ggt gga gac aga gat aaa atc act ata 576
Tyr Asp Asn Ile Glu Lys Phe Gly Gly Asp Arg Asp Lys Ile Thr Ile 180
185 190 gct gga gaa tct gct gga gca gca agt gtt cat ttt ctg atg atg
gac 624 Ala Gly Glu Ser Ala Gly Ala Ala Ser Val His Phe Leu Met Met
Asp 195 200 205 aat tct act aga aaa tac tac caa agg gca att ttg cag
agt ggg aca 672 Asn Ser Thr Arg Lys Tyr Tyr Gln Arg Ala Ile Leu Gln
Ser Gly Thr 210 215 220 tta ctc aat ccg act gct aat caa att caa cct
ctg cat aga ttt gaa 720 Leu Leu Asn Pro Thr Ala Asn Gln Ile Gln Pro
Leu His Arg Phe Glu 225 230 235 240 aaa cta aaa caa gtg ctg aac atc
acg caa aaa caa gaa ctc cta aat 768 Lys Leu Lys Gln Val Leu Asn Ile
Thr Gln Lys Gln Glu Leu Leu Asn 245 250 255 ctg gac aaa aat caa att
ttg cga gca gcc tta aac aga gtc cca gat 816 Leu Asp Lys Asn Gln Ile
Leu Arg Ala Ala Leu Asn Arg Val Pro Asp 260 265 270 aac aac gac cac
gaa agg gac aca gta cca gta ttt aat cca gtc cta 864 Asn Asn Asp His
Glu Arg Asp Thr Val Pro Val Phe Asn Pro Val Leu 275 280 285 gaa tca
cca gaa tct cca gac cca ata aca ttt cca tct gct tta gaa 912 Glu Ser
Pro Glu Ser Pro Asp Pro Ile Thr Phe Pro Ser Ala Leu Glu 290 295 300
aga atg aga aat ggt gaa ttt cct gac gtt gat gtc atc att gga ttc 960
Arg Met Arg Asn Gly Glu Phe Pro Asp Val Asp Val Ile Ile Gly Phe 305
310 315 320 aat agt gct gaa ggt tta aga tct atg cca aga gta acc aga
gga aac 1008 Asn Ser Ala Glu Gly Leu Arg Ser Met Pro Arg Val Thr
Arg Gly Asn 325 330 335 atg gaa gtt tac aag act ttg aca aat ata gag
aga gct ata cct aga 1056 Met Glu Val Tyr Lys Thr Leu Thr Asn Ile
Glu Arg Ala Ile Pro Arg 340 345 350 gat gct aat att tgg aaa aat cct
aat ggc att gag gag aaa aaa ctt 1104 Asp Ala Asn Ile Trp Lys Asn
Pro Asn Gly Ile Glu Glu Lys Lys Leu 355 360 365 ata aaa atg ctt aca
gag ttt tat gac caa gtt aaa gaa caa aac gat 1152 Ile Lys Met Leu
Thr Glu Phe Tyr Asp Gln Val Lys Glu Gln Asn Asp 370 375 380 gac atc
gaa gcc tat gtc caa cta aaa ggc gat gct ggt tat ctc caa 1200 Asp
Ile Glu Ala Tyr Val Gln Leu Lys Gly Asp Ala Gly Tyr Leu Gln 385 390
395 400 gga att tac cgt acc ttg aaa gcc ata ttt ttc aat gaa atc aaa
aga 1248 Gly Ile Tyr Arg Thr Leu Lys Ala Ile Phe Phe Asn Glu Ile
Lys Arg 405 410 415 aat tcc aac ttg tat ttg tat agg tta tca gat gat
acg tat agt gta 1296 Asn Ser Asn Leu Tyr Leu Tyr Arg Leu Ser Asp
Asp Thr Tyr Ser Val 420 425 430 tat aaa agt tat atc ttg ccc tat cga
tgg ggt tcc ttg cca gga gtt 1344 Tyr Lys Ser Tyr Ile Leu Pro Tyr
Arg Trp Gly Ser Leu Pro Gly Val 435 440 445 agt cat ggt gat gat tta
gga tat ctt ttt gca aac tct ttg gat gtt 1392 Ser His Gly Asp Asp
Leu Gly Tyr Leu Phe Ala Asn Ser Leu Asp Val 450 455 460 cct att ttg
gga aca acg cac att tct ata ccg caa gat gct atg cag 1440 Pro Ile
Leu Gly Thr Thr His Ile Ser Ile Pro Gln Asp Ala Met Gln 465 470 475
480 act ctg gaa agg atg gtc agg atc tgg acc aat ttt gta aag aat gga
1488 Thr Leu Glu Arg Met Val Arg Ile Trp Thr Asn Phe Val Lys Asn
Gly 485 490 495 aaa cct aca tca aac act gaa gat gca tca tgt gat aca
aaa aga cat 1536 Lys Pro Thr Ser Asn Thr Glu Asp Ala Ser Cys Asp
Thr Lys Arg His 500 505 510 tta aac gac att ttt tgg gaa cca tac aac
gac gaa gaa cca aaa tat 1584 Leu Asn Asp Ile Phe Trp Glu Pro Tyr
Asn Asp Glu Glu Pro Lys Tyr 515 520 525 ttg gac atg gga aaa gaa cat
ttt gaa atg aaa aat att ttg gaa cta 1632 Leu Asp Met Gly Lys Glu
His Phe Glu Met Lys Asn Ile Leu Glu Leu 530 535 540 aaa cgc atg atg
ctt tgg gat gaa gtt tat aga aat gcg aat ttg cgg 1680 Lys Arg Met
Met Leu Trp Asp Glu Val Tyr Arg Asn Ala Asn Leu Arg 545 550 555 560
ttt aga gtc tgt aat gaa gaa agt att aga 1710 Phe Arg Val Cys Asn
Glu Glu Ser Ile Arg 565 570 34 1785 DNA Ctenocephalides felis exon
(1)..(1785) 34 atg tta cct cac agt gca tta gtt tta ttt tta ttt ttt
tta ttt ttc 48 Met Leu Pro His Ser Ala Leu Val Leu Phe Leu Phe Phe
Leu Phe Phe 1 5 10 15 tta ttt aca cct gtc ttg tgc ata cta tgg gat
aac cta gat cag cat 96 Leu Phe Thr Pro Val Leu Cys Ile Leu Trp Asp
Asn Leu Asp Gln His 20 25 30 ttg tgc aga gtt caa ttt aac ggg atc
acg gaa gga aaa ccg ttc cga 144 Leu Cys Arg Val Gln Phe Asn Gly Ile
Thr Glu Gly Lys Pro Phe Arg 35 40 45 tat aaa gat cat aaa aat gat
gta tat tgt tcc tat ttg gga att cct 192 Tyr Lys Asp His Lys Asn Asp
Val Tyr Cys Ser Tyr Leu Gly Ile Pro 50 55 60 tat gca gaa ccg cct
att gga cca ttg cga ttt cag tct cca aaa cca 240 Tyr Ala Glu Pro Pro
Ile Gly Pro Leu Arg Phe Gln Ser Pro Lys Pro 65 70 75 80 ata tca aat
cca aaa aca gga ttc gtt cag gct cgg tct tta gga gac 288 Ile Ser Asn
Pro Lys Thr Gly Phe Val Gln Ala Arg Ser Leu Gly Asp 85 90 95 aaa
tgt ttc cag gaa agt cta ata tat tct tat gca gga agc gaa gat 336 Lys
Cys Phe Gln Glu Ser Leu Ile Tyr Ser Tyr Ala Gly Ser Glu Asp 100 105
110 tgc tta tat ctg aat ata ttc acg cca gag act gtt aat tct gcg aac
384 Cys Leu Tyr Leu Asn Ile Phe Thr Pro Glu Thr Val Asn Ser Ala Asn
115 120 125 aat aca aaa tat cct gta atg ttc tgg atc cat gga ggc gca
ttc aac 432 Asn Thr Lys Tyr Pro Val Met Phe Trp Ile His Gly Gly Ala
Phe Asn 130 135 140 caa gga tca gga tct tat aat ttt ttt gga cct gat
tat ttg atc agg 480 Gln Gly Ser Gly Ser Tyr Asn Phe Phe Gly Pro Asp
Tyr Leu Ile Arg 145 150 155 160 gaa gga att att ttg gtc act atc aac
tat aga tta gga gtt ttc ggt 528 Glu Gly Ile Ile Leu Val Thr Ile Asn
Tyr Arg Leu Gly Val Phe Gly 165 170 175 ttt cta tca gcg ccg gaa tgg
gat atc cat gga aat atg ggt cta aaa 576 Phe Leu Ser Ala Pro Glu Trp
Asp Ile His Gly Asn Met Gly Leu Lys 180 185 190 gac cag aga ttg gca
cta aaa tgg gtt tat gac aac atc gaa aaa ttt 624 Asp Gln Arg Leu Ala
Leu Lys Trp Val Tyr Asp Asn Ile Glu Lys Phe 195 200 205 ggt gga gac
aga gat aaa atc act ata gct gga gaa tct gct gga gca 672 Gly Gly Asp
Arg Asp Lys Ile Thr Ile Ala Gly Glu Ser Ala Gly Ala 210 215 220 gca
agt gtt cat ttt ctg atg atg gac aat tct act aga aaa tac tac 720 Ala
Ser Val His Phe Leu Met Met Asp Asn Ser Thr Arg Lys Tyr Tyr 225 230
235 240 caa agg gca att ttg cag agt ggg aca tta ctc aat ccg act gct
aat 768 Gln Arg Ala Ile Leu Gln Ser Gly Thr Leu Leu Asn Pro Thr Ala
Asn 245 250 255 caa att caa cct ctg cat aga ttt gaa aaa cta aaa caa
gtg ctg aac 816 Gln Ile Gln Pro Leu His Arg Phe Glu Lys Leu Lys Gln
Val Leu Asn 260 265 270 atc acg caa aaa caa gaa ctc cta aat ctg gac
aaa aat caa att ttg 864 Ile Thr Gln Lys Gln Glu Leu Leu Asn Leu Asp
Lys Asn Gln Ile Leu 275 280 285 cga gca gcc tta aac aga gtc cca gat
aac aac gac cac gaa agg gac 912 Arg Ala Ala Leu Asn Arg Val Pro Asp
Asn Asn Asp His Glu Arg Asp 290 295 300 aca gta cca gta ttt aat cca
gtc cta gaa tca cca gaa tct cca gac 960 Thr Val Pro Val Phe Asn Pro
Val Leu Glu Ser Pro Glu Ser Pro Asp 305 310 315 320 cca ata aca ttt
cca tct gct tta gaa aga atg aga aat ggt gaa ttt 1008 Pro Ile Thr
Phe Pro Ser Ala Leu Glu Arg Met Arg Asn Gly Glu Phe 325 330 335 cct
gac gtt gat gtc atc att gga ttc aat agt gct gaa ggt tta aga 1056
Pro Asp Val Asp Val Ile Ile Gly Phe Asn Ser Ala Glu Gly Leu Arg 340
345 350 tct atg cca aga gta acc aga gga aac atg gaa gtt tac aag act
ttg 1104 Ser Met Pro Arg Val Thr Arg Gly Asn Met Glu Val Tyr Lys
Thr Leu 355 360 365 aca aat ata gag aga gct ata cct aga gat gct aat
att tgg aaa aat 1152 Thr Asn Ile Glu Arg Ala Ile Pro Arg Asp Ala
Asn Ile Trp Lys Asn 370 375 380 cct aat ggc att gag gag aaa aaa ctt
ata aaa atg ctt aca gag ttt 1200 Pro Asn Gly Ile Glu Glu Lys Lys
Leu Ile Lys Met Leu Thr Glu Phe 385 390 395 400 tat gac caa gtt aaa
gaa caa aac gat gac atc gaa gcc tat gtc caa 1248 Tyr Asp Gln Val
Lys Glu Gln Asn Asp Asp Ile Glu Ala Tyr Val Gln 405 410 415 cta aaa
ggc gat gct ggt tat ctc caa gga att tac cgt acc ttg aaa 1296 Leu
Lys Gly Asp Ala Gly Tyr Leu Gln Gly Ile Tyr Arg Thr Leu Lys 420 425
430 gcc ata ttt ttc aat gaa atc aaa aga aat tcc aac ttg tat ttg tat
1344 Ala Ile Phe Phe Asn Glu Ile Lys Arg Asn Ser Asn Leu Tyr Leu
Tyr 435 440 445 agg tta tca gat gat acg tat agt gta tat aaa agt tat
atc ttg ccc 1392 Arg Leu Ser Asp Asp Thr Tyr Ser Val Tyr Lys Ser
Tyr Ile Leu Pro 450 455 460 tat cga tgg ggt tcc ttg cca gga gtt agt
cat ggt gat gat tta gga 1440 Tyr Arg Trp Gly Ser Leu Pro Gly Val
Ser His Gly Asp Asp Leu Gly 465 470 475 480 tat ctt ttt gca aac tct
ttg gat gtt cct att ttg gga aca acg cac 1488 Tyr Leu Phe Ala Asn
Ser Leu Asp Val Pro Ile Leu Gly Thr Thr His 485 490 495 att tct ata
ccg caa gat gct atg cag act ctg gaa agg atg gtc agg 1536 Ile Ser
Ile Pro Gln Asp Ala Met Gln Thr Leu Glu Arg Met Val Arg 500 505 510
atc tgg acc aat ttt gta aag aat gga aaa cct aca tca aac act gaa
1584 Ile Trp Thr Asn Phe Val Lys Asn Gly Lys Pro Thr Ser Asn Thr
Glu 515 520 525 gat gca tca tgt gat aca aaa aga cat tta aac gac att
ttt tgg gaa 1632 Asp Ala Ser Cys Asp Thr Lys Arg His Leu Asn Asp
Ile Phe Trp Glu 530 535 540 cca tac aac gac gaa gaa cca aaa tat ttg
gac atg gga aaa gaa cat 1680 Pro Tyr Asn Asp Glu Glu Pro Lys Tyr
Leu Asp Met Gly Lys Glu His 545 550 555 560 ttt gaa atg aaa aat att
ttg gaa cta aaa cgc atg atg ctt tgg gat 1728 Phe Glu Met Lys Asn
Ile Leu Glu Leu Lys Arg Met Met Leu Trp Asp 565 570 575 gaa gtt tat
aga aat gcg aat ttg cgg ttt aga gtc tgt aat gaa gaa 1776 Glu Val
Tyr Arg Asn Ala Asn Leu Arg Phe Arg Val Cys Asn Glu Glu 580 585 590
agt att aga 1785 Ser Ile Arg 595 35 1785 DNA Ctenocephalides felis
35 tctaatactt tcttcattac agactctaaa ccgcaaattc gcatttctat
aaacttcatc 60 ccaaagcatc atgcgtttta gttccaaaat atttttcatt
tcaaaatgtt cttttcccat 120 gtccaaatat tttggttctt cgtcgttgta
tggttcccaa aaaatgtcgt ttaaatgtct 180 ttttgtatca catgatgcat
cttcagtgtt tgatgtaggt tttccattct ttacaaaatt 240 ggtccagatc
ctgaccatcc tttccagagt ctgcatagca tcttgcggta tagaaatgtg 300
cgttgttccc aaaataggaa catccaaaga gtttgcaaaa agatatccta aatcatcacc
360 atgactaact cctggcaagg aaccccatcg atagggcaag atataacttt
tatatacact 420 atacgtatca tctgataacc tatacaaata caagttggaa
tttcttttga tttcattgaa 480 aaatatggct ttcaaggtac ggtaaattcc
ttggagataa ccagcatcgc cttttagttg 540 gacataggct tcgatgtcat
cgttttgttc tttaacttgg tcataaaact ctgtaagcat 600 ttttataagt
tttttctcct caatgccatt aggatttttc caaatattag catctctagg 660
tatagctctc tctatatttg tcaaagtctt gtaaacttcc atgtttcctc tggttactct
720 tggcatagat cttaaacctt cagcactatt gaatccaatg atgacatcaa
cgtcaggaaa 780 ttcaccattt ctcattcttt ctaaagcaga tggaaatgtt
attgggtctg gagattctgg 840 tgattctagg actggattaa atactggtac
tgtgtccctt tcgtggtcgt tgttatctgg 900 gactctgttt aaggctgctc
gcaaaatttg atttttgtcc agatttagga gttcttgttt 960 ttgcgtgatg
ttcagcactt gttttagttt ttcaaatcta tgcagaggtt gaatttgatt 1020
agcagtcgga ttgagtaatg tcccactctg caaaattgcc ctttggtagt attttctagt
1080 agaattgtcc atcatcagaa aatgaacact tgctgctcca gcagattctc
cagctatagt 1140 gattttatct ctgtctccac caaatttttc gatgttgtca
taaacccatt ttagtgccaa 1200 tctctggtct tttagaccca tatttccatg
gatatcccat tccggcgctg atagaaaacc 1260 gaaaactcct aatctatagt
tgatagtgac caaaataatt ccttccctga tcaaataatc 1320 aggtccaaaa
aaattataag atcctgatcc ttggttgaat gcgcctccat ggatccagaa 1380
cattacagga tattttgtat tgttcgcaga attaacagtc tctggcgtga atatattcag
1440 atataagcaa tcttcgcttc ctgcataaga atatattaga ctttcctgga
aacatttgtc 1500 tcctaaagac cgagcctgaa cgaatcctgt ttttggattt
gatattggtt ttggagactg 1560 aaatcgcaat ggtccaatag gcggttctgc
ataaggaatt cccaaatagg aacaatatac 1620 atcattttta tgatctttat
atcggaacgg ttttccttcc gtgatcccgt taaattgaac 1680 tctgcacaaa
tgctgatcta ggttatccca tagtatgcac aagacaggtg taaataagaa 1740
aaataaaaaa aataaaaata aaactaatgc actgtgaggt aacat 1785 36 2007 DNA
Ctenocephalides felis CDS (11)..(1594) 36 agttccaacg atg gct gat
cta caa gtg act ttg ctt caa ggt act tta 49 Met Ala Asp Leu Gln Val
Thr Leu Leu Gln Gly Thr Leu 1 5 10 aaa gga aaa gag caa att agt gaa
aaa gga aat gtg ttc cat agt tat 97 Lys Gly Lys Glu Gln Ile Ser Glu
Lys Gly Asn Val Phe His Ser Tyr 15 20 25 tct gga att cca tat gcc
aaa cct cct gta ggt gat cta aga ttt aag 145 Ser Gly Ile Pro Tyr Ala
Lys Pro Pro Val Gly Asp Leu Arg Phe Lys 30 35 40 45 cca cct caa cct
gca gaa cct tgg tca ggt gtt ctt gat gct agt aaa 193 Pro Pro Gln Pro
Ala Glu Pro Trp Ser Gly Val Leu Asp Ala Ser Lys 50 55 60 gaa ggg
aat agt tgt aga tca gta cat ttt att aaa aaa att aaa gta 241 Glu Gly
Asn Ser Cys Arg Ser Val His Phe Ile Lys Lys Ile Lys Val 65 70 75
ggg gct gaa gat tgt tta tac ctc aat gtc tat gta cca aaa aca tca 289
Gly Ala Glu Asp Cys Leu Tyr Leu Asn Val Tyr Val Pro Lys Thr Ser 80
85 90 gag aaa tca ctt ctt cca gta atg gta tgg ata cat gga gga ggc
ttc 337 Glu Lys Ser Leu Leu Pro Val Met Val Trp Ile His Gly Gly Gly
Phe 95 100 105 ttc atg gga tct gga aat agt gat atg tat ggt cct gaa
tat ttg atg 385 Phe Met Gly Ser Gly Asn Ser Asp Met Tyr Gly Pro Glu
Tyr Leu Met 110 115 120 125 gat tat gga att gtt ctg gtt act ttc aat
tat cga tta ggt gtt ttg 433 Asp Tyr Gly Ile Val Leu Val Thr Phe Asn
Tyr Arg Leu Gly Val Leu 130 135 140 gga ttt ttg aac ctg gga ata gaa
gaa gcg cct ggc aat gtt ggt ttg 481 Gly Phe Leu Asn Leu Gly Ile Glu
Glu Ala Pro Gly Asn Val Gly Leu 145 150 155 atg gac cag gtt gaa gct
cta aaa tgg gta aaa aac aat att gca tcc 529 Met Asp Gln Val Glu Ala
Leu Lys Trp Val Lys Asn Asn Ile Ala Ser 160 165 170 ttt ggt ggt gac
ccc aac aat gtg act att ttt gga gaa tca gca ggt 577 Phe Gly Gly Asp
Pro Asn Asn Val Thr Ile Phe Gly Glu Ser Ala Gly 175 180 185 ggt gca
agt gtt cat tat ttg atg tta tca gat ctt tcc aaa gga ctt 625 Gly Ala
Ser Val His Tyr Leu Met Leu Ser Asp Leu Ser Lys Gly Leu 190 195 200
205 ttt cat aaa gcg atc tca caa agt gga agt gct ttt aat cct tgg gca
673 Phe His Lys Ala Ile Ser Gln Ser Gly Ser Ala Phe Asn Pro Trp Ala
210 215 220 ctt caa cat gat aat aat aaa gaa aat gca ttc cgc ctc tgc
aaa ctt 721 Leu Gln His Asp Asn Asn Lys Glu Asn Ala Phe Arg Leu Cys
Lys Leu 225 230 235 ctg ggt cat cct gtc gat aac gag aca gaa gct cta
aaa atc ctt cgt 769 Leu Gly His Pro Val Asp Asn Glu Thr Glu Ala Leu
Lys Ile Leu Arg 240 245 250 caa gcc ccc ata gat gat ctt ata gac aac
aga ata aaa cca aaa gac 817 Gln Ala Pro Ile Asp Asp Leu Ile Asp Asn
Arg Ile Lys Pro Lys Asp 255 260 265 aaa ggc caa ctt att ata gac tat
cct ttt cta cca aca ata gaa aaa 865 Lys Gly Gln Leu Ile Ile Asp Tyr
Pro Phe Leu Pro Thr Ile Glu Lys 270 275 280 285 cgt tat caa aat ttt
gaa cca ttc ttg gac cag tct cca tta tca aaa 913 Arg Tyr Gln Asn Phe
Glu Pro Phe Leu Asp Gln Ser Pro Leu Ser Lys 290 295 300 atg caa tca
ggc aat ttc aca aaa gtc cca ttt ata tgt gga tac aac 961 Met Gln Ser
Gly Asn Phe Thr Lys Val Pro Phe Ile Cys Gly Tyr Asn 305 310 315 agt
gct gaa gga att tta ggt tta atg gac ttc aag gat gac cca aat 1009
Ser Ala Glu Gly Ile Leu Gly Leu Met Asp Phe Lys Asp Asp Pro Asn 320
325 330 ata ttt gag aag ttt gaa gct gat ttt gaa aga ttt gta cca gta
gat 1057 Ile Phe Glu Lys Phe Glu Ala Asp Phe Glu Arg Phe Val Pro
Val Asp 335 340 345 ttg aat cta act tta agg tct aag gaa tct aaa aaa
ttg gct gaa gaa 1105 Leu Asn Leu Thr Leu Arg Ser Lys Glu Ser Lys
Lys Leu Ala Glu Glu 350 355 360 365 atg aga aag ttt tat tac caa gac
gaa cct gtt tct tca gac aac aaa 1153 Met Arg Lys Phe Tyr Tyr Gln
Asp Glu Pro Val Ser Ser Asp Asn Lys 370 375 380 gaa aaa ttt gtc agt
gtt att agt gat act tgg ttt ttg aga ggg att 1201 Glu Lys Phe Val
Ser Val Ile Ser Asp Thr Trp Phe Leu Arg Gly Ile 385 390 395 aaa aat
act gca aga tat ata att gaa cat tcc tca gaa ccg tta tat 1249 Lys
Asn Thr Ala Arg Tyr Ile Ile Glu His Ser Ser Glu Pro Leu Tyr 400 405
410 tta tat gtt tat agt ttt gat gat ttt ggt ttt ttg aag aaa ctt gta
1297 Leu Tyr Val Tyr Ser Phe Asp Asp Phe Gly Phe Leu Lys Lys Leu
Val 415 420 425 tta gat cct aat att gaa gga gca gct cat gga gat gag
ctg gga tat 1345 Leu Asp Pro Asn Ile Glu Gly Ala Ala His Gly Asp
Glu Leu Gly Tyr 430 435 440 445 ctt ttc aag atg agt ttt aca gaa ttt
cca aaa gat tta cca agt gca 1393 Leu Phe Lys Met Ser Phe Thr Glu
Phe Pro Lys Asp Leu Pro Ser Ala 450 455 460 gtg gtg aat agg gaa cga
ttg ttg caa ctt tgg aca aat ttt gca aaa 1441 Val Val Asn Arg Glu
Arg Leu Leu Gln Leu Trp Thr Asn Phe Ala Lys 465 470 475 aca gga aat
ccc act cct gaa atc aat gat gtt ata aca aca aaa tgg 1489 Thr Gly
Asn Pro Thr Pro Glu Ile Asn Asp Val Ile Thr Thr Lys Trp 480 485 490
gat aaa gct act gag gaa aaa tca gat cat atg gat atc gat aat act
1537 Asp Lys Ala Thr Glu Glu Lys Ser Asp His Met Asp Ile Asp Asn
Thr 495 500 505 ttg aga atg att cca gat cct gat gca aaa cga ctt aga
ttt tgg aat 1585 Leu Arg Met Ile Pro Asp Pro Asp Ala Lys Arg Leu
Arg Phe Trp Asn 510 515 520 525 aaa ttt tta tgataaatat accaattatc
gattttatta tagagtttct 1634 Lys Phe Leu gtattagtat aattatcacg
tttagatgta cgagattcaa ttggctctaa ttgaagtata 1694 tttcgatttc
aaatttactc tgattattgg aaaaaaagct tttacagttg taataatcaa 1754
gaagtaggtg gtaaatttag aacaaattct gttttagtga tttgcgcatt caacagatgg
1814 tgtactgtgc ctaaatttgt cgctcttctt gaagaactga actaaaaatg
tgattaatgg 1874 acgccacatt atttatattt gatattatta ccatctttgt
atcatatttg cttttatttt 1934 ttcatttttt ttttatttca aatatattgt
ttttttataa aaaaaaaaaa aaaaaaaaaa 1994 aaaaaaaaaa aaa 2007 37 528
PRT Ctenocephalides felis 37 Met Ala Asp Leu Gln Val Thr Leu Leu
Gln Gly Thr Leu Lys Gly Lys 1 5 10 15 Glu Gln Ile Ser Glu Lys Gly
Asn Val Phe His Ser Tyr Ser Gly Ile 20 25 30 Pro Tyr Ala Lys Pro
Pro Val Gly Asp Leu Arg Phe Lys Pro Pro Gln 35 40 45 Pro Ala Glu
Pro Trp Ser Gly Val Leu Asp Ala Ser Lys Glu Gly Asn 50 55 60 Ser
Cys Arg Ser Val His Phe Ile Lys Lys Ile Lys Val Gly Ala Glu 65 70
75 80 Asp Cys Leu Tyr Leu Asn Val Tyr Val Pro Lys Thr Ser Glu Lys
Ser 85 90 95 Leu Leu Pro Val Met Val Trp Ile His Gly Gly Gly Phe
Phe Met Gly 100 105 110 Ser Gly Asn Ser Asp Met Tyr Gly Pro Glu Tyr
Leu Met Asp Tyr Gly 115 120 125 Ile Val Leu Val Thr Phe Asn Tyr Arg
Leu Gly Val Leu Gly Phe Leu 130 135 140 Asn Leu Gly Ile Glu Glu Ala
Pro Gly Asn Val Gly Leu Met Asp Gln 145 150 155 160 Val Glu Ala Leu
Lys Trp Val Lys Asn Asn Ile Ala Ser Phe Gly Gly 165 170 175 Asp Pro
Asn Asn Val Thr Ile Phe Gly Glu Ser Ala Gly Gly Ala Ser 180 185 190
Val His Tyr Leu Met Leu Ser Asp Leu Ser Lys Gly Leu Phe His Lys 195
200 205 Ala Ile Ser Gln Ser Gly Ser Ala Phe Asn Pro Trp Ala Leu Gln
His 210 215 220 Asp Asn Asn Lys Glu Asn Ala Phe Arg Leu Cys Lys Leu
Leu Gly His 225 230 235 240 Pro Val Asp Asn Glu Thr Glu Ala Leu Lys
Ile Leu Arg Gln Ala Pro 245 250 255 Ile Asp Asp Leu Ile Asp Asn Arg
Ile Lys Pro Lys Asp Lys Gly Gln 260 265 270 Leu Ile Ile Asp Tyr Pro
Phe Leu Pro Thr Ile Glu Lys Arg Tyr Gln 275 280 285 Asn Phe Glu Pro
Phe Leu Asp Gln Ser Pro Leu Ser Lys Met Gln Ser 290 295 300 Gly Asn
Phe Thr Lys Val Pro Phe Ile Cys Gly Tyr Asn Ser Ala Glu 305 310 315
320 Gly Ile Leu Gly Leu Met Asp Phe Lys Asp Asp Pro Asn Ile Phe Glu
325 330 335 Lys Phe Glu Ala Asp Phe Glu Arg Phe Val Pro Val Asp Leu
Asn Leu 340 345 350 Thr Leu Arg Ser Lys Glu Ser Lys Lys Leu Ala Glu
Glu Met Arg Lys 355 360 365 Phe Tyr Tyr Gln Asp Glu Pro Val Ser Ser
Asp Asn Lys Glu Lys Phe 370 375 380 Val Ser Val Ile Ser Asp Thr Trp
Phe Leu Arg Gly Ile Lys Asn Thr 385 390 395 400 Ala Arg Tyr Ile Ile
Glu His Ser Ser Glu Pro Leu Tyr Leu Tyr Val 405 410 415 Tyr Ser Phe
Asp Asp Phe Gly Phe Leu Lys Lys Leu Val Leu Asp Pro 420 425 430 Asn
Ile Glu Gly Ala Ala His Gly Asp Glu Leu Gly Tyr Leu Phe Lys 435 440
445 Met Ser Phe Thr Glu Phe Pro Lys Asp Leu Pro Ser Ala Val Val Asn
450 455 460 Arg Glu Arg Leu Leu Gln Leu Trp Thr Asn Phe Ala Lys Thr
Gly Asn 465 470 475 480 Pro Thr Pro Glu Ile Asn Asp Val Ile Thr Thr
Lys Trp Asp Lys Ala 485 490 495 Thr Glu Glu Lys Ser Asp His Met Asp
Ile Asp Asn Thr Leu Arg Met 500 505 510 Ile Pro Asp Pro Asp Ala Lys
Arg Leu Arg Phe Trp Asn Lys Phe Leu 515 520 525 38 2007 DNA
Ctenocephalides felis 38 tttttttttt tttttttttt tttttttttt
tttttataaa aaaacaatat atttgaaata 60 aaaaaaaaat gaaaaaataa
aagcaaatat gatacaaaga tggtaataat atcaaatata 120 aataatgtgg
cgtccattaa tcacattttt agttcagttc ttcaagaaga gcgacaaatt 180
taggcacagt acaccatctg ttgaatgcgc aaatcactaa aacagaattt gttctaaatt
240 taccacctac ttcttgatta ttacaactgt aaaagctttt tttccaataa
tcagagtaaa 300 tttgaaatcg aaatatactt caattagagc caattgaatc
tcgtacatct aaacgtgata 360 attatactaa tacagaaact ctataataaa
atcgataatt ggtatattta tcataaaaat 420 ttattccaaa atctaagtcg
ttttgcatca ggatctggaa tcattctcaa agtattatcg 480 atatccatat
gatctgattt ttcctcagta gctttatccc attttgttgt tataacatca 540
ttgatttcag gagtgggatt tcctgttttt gcaaaatttg tccaaagttg caacaatcgt
600 tccctattca ccactgcact tggtaaatct tttggaaatt ctgtaaaact
catcttgaaa 660 agatatccca gctcatctcc atgagctgct ccttcaatat
taggatctaa tacaagtttc 720 ttcaaaaaac caaaatcatc aaaactataa
acatataaat ataacggttc tgaggaatgt 780 tcaattatat atcttgcagt
atttttaatc cctctcaaaa accaagtatc actaataaca 840 ctgacaaatt
tttctttgtt gtctgaagaa acaggttcgt cttggtaata aaactttctc 900
atttcttcag ccaatttttt agattcctta gaccttaaag ttagattcaa atctactggt
960 acaaatcttt caaaatcagc ttcaaacttc tcaaatatat ttgggtcatc
cttgaagtcc 1020 attaaaccta aaattccttc agcactgttg tatccacata
taaatgggac ttttgtgaaa 1080 ttgcctgatt gcatttttga taatggagac
tggtccaaga atggttcaaa attttgataa 1140 cgtttttcta ttgttggtag
aaaaggatag tctataataa gttggccttt gtcttttggt 1200 tttattctgt
tgtctataag atcatctatg ggggcttgac gaaggatttt tagagcttct 1260
gtctcgttat cgacaggatg acccagaagt ttgcagaggc ggaatgcatt ttctttatta
1320 ttatcatgtt gaagtgccca aggattaaaa gcacttccac tttgtgagat
cgctttatga 1380 aaaagtcctt tggaaagatc tgataacatc aaataatgaa
cacttgcacc acctgctgat 1440 tctccaaaaa tagtcacatt gttggggtca
ccaccaaagg atgcaatatt gttttttacc 1500 cattttagag cttcaacctg
gtccatcaaa ccaacattgc caggcgcttc ttctattccc 1560 aggttcaaaa
atcccaaaac acctaatcga taattgaaag taaccagaac aattccataa 1620
tccatcaaat attcaggacc atacatatca ctatttccag atcccatgaa gaagcctcct
1680 ccatgtatcc ataccattac tggaagaagt gatttctctg atgtttttgg
tacatagaca 1740 ttgaggtata aacaatcttc agcccctact ttaatttttt
taataaaatg tactgatcta 1800 caactattcc cttctttact agcatcaaga
acacctgacc aaggttctgc aggttgaggt 1860 ggcttaaatc ttagatcacc
tacaggaggt ttggcatatg gaattccaga ataactatgg 1920 aacacatttc
ctttttcact aatttgctct tttcctttta aagtaccttg aagcaaagtc 1980
acttgtagat cagccatcgt tggaact 2007 39 12 PRT Peptide 39 Asp Pro Pro
Thr Val Thr Leu Pro Gln Gly Glu Leu 1 5 10 40 22 PRT Peptide
MISC_FEATURE (21)..(21) Xaa = unknown 40 Asp Pro Pro Thr Val Thr
Leu Pro Gln Gly Glu Leu Val Gly Lys Ala 1 5 10 15 Thr Asn Glu Asn
Xaa Lys 20 41 12 PRT Peptide 41 Asp Pro Pro Thr Val Thr Leu Pro Gln
Gly Glu Leu 1 5 10 42 21 PRT Peptide 42 Asp Pro Pro Thr Val Thr Leu
Pro Gln Gly Glu Leu Val Gly Lys Ala 1 5 10 15 Leu Ser Asn Glu Asn
20 43 8 PRT Peptide 43 Asp Pro Pro Thr Val Thr Leu Pro 1 5 44 23
PRT Peptide 44 Asp Pro Pro Thr Val Thr Leu Pro Gln Gly Glu Leu Val
Gly Lys Ala 1 5 10 15 Leu Thr Asn Glu Asn Gly Lys 20 45 20 DNA
Artificial sequence Synthetic Primer 45 aattaaccct cactaaaggg 20 46
17 DNA Artificial sequence Synthetic Primer 46 ardccdccdc crtrdat
17 47 38 DNA Artificial sequence Synthetic Primer 47 tgtgctcgag
atgggataac ctagatcagc atttgtgc 38 48 35 DNA Artificial sequence
Synthetic Primer 48 ttaaggtacc tcatctaata cttccttcat tacag 35 49 36
DNA Artificial sequence Synthetic Primer 49 aaaactgcag tataaatatg
ttacctcaca gtagtg 36 50 34 DNA Artificial sequence Synthetic Primer
50 tgctctagat tatctaatac ttccttcatt acag 34 51 1584 DNA
Ctenocephalides felis exon (1)..(1584) 51 atg gct gat cta caa gtg
act ttg ctt caa ggt act tta aaa gga aaa 48 Met Ala Asp Leu Gln Val
Thr Leu Leu Gln Gly Thr Leu Lys Gly Lys 1 5 10 15 gag caa att agt
gaa aaa gga aat gtg ttc cat agt tat tct gga att 96 Glu Gln Ile Ser
Glu Lys Gly Asn Val Phe His Ser Tyr Ser Gly Ile 20 25 30 cca tat
gcc aaa cct cct gta ggt gat cta aga ttt aag cca cct caa 144 Pro Tyr
Ala Lys Pro Pro Val Gly Asp Leu Arg Phe Lys Pro Pro Gln 35 40 45
cct gca gaa cct tgg tca ggt gtt ctt gat gct agt aaa gaa ggg aat 192
Pro Ala Glu Pro Trp Ser Gly Val Leu Asp Ala Ser Lys Glu Gly Asn 50
55 60 agt tgt aga tca gta cat ttt att aaa aaa att aaa gta ggg gct
gaa 240 Ser Cys Arg Ser Val His Phe Ile Lys Lys Ile Lys Val Gly Ala
Glu 65 70 75 80 gat tgt tta tac ctc aat gtc tat gta cca aaa aca tca
gag aaa tca 288 Asp Cys Leu Tyr Leu Asn Val Tyr Val Pro Lys Thr Ser
Glu Lys Ser 85 90 95 ctt ctt cca gta atg gta tgg ata cat gga gga
ggc ttc ttc atg gga 336 Leu Leu Pro Val Met Val Trp Ile His Gly Gly
Gly Phe Phe Met Gly 100 105 110 tct gga aat agt gat atg tat ggt cct
gaa tat ttg atg gat tat gga 384 Ser Gly Asn Ser Asp Met Tyr Gly Pro
Glu Tyr Leu Met Asp Tyr Gly 115 120 125 att gtt ctg gtt act ttc aat
tat cga tta ggt gtt ttg gga ttt ttg 432 Ile Val Leu Val Thr Phe Asn
Tyr Arg Leu Gly Val Leu Gly Phe Leu 130 135 140 aac ctg gga ata gaa
gaa gcg cct ggc aat gtt ggt ttg atg gac cag 480 Asn Leu Gly Ile Glu
Glu Ala Pro Gly Asn Val Gly Leu Met Asp Gln 145 150 155 160 gtt gaa
gct cta aaa tgg gta aaa aac aat att gca tcc ttt ggt ggt 528 Val Glu
Ala Leu Lys Trp Val Lys Asn Asn Ile Ala Ser Phe Gly Gly 165 170 175
gac ccc aac aat gtg act att ttt gga gaa tca gca ggt ggt gca agt 576
Asp Pro Asn Asn Val Thr Ile Phe Gly Glu Ser Ala Gly Gly Ala Ser 180
185 190 gtt cat tat ttg atg tta tca gat ctt tcc aaa
gga ctt ttt cat aaa 624 Val His Tyr Leu Met Leu Ser Asp Leu Ser Lys
Gly Leu Phe His Lys 195 200 205 gcg atc tca caa agt gga agt gct ttt
aat cct tgg gca ctt caa cat 672 Ala Ile Ser Gln Ser Gly Ser Ala Phe
Asn Pro Trp Ala Leu Gln His 210 215 220 gat aat aat aaa gaa aat gca
ttc cgc ctc tgc aaa ctt ctg ggt cat 720 Asp Asn Asn Lys Glu Asn Ala
Phe Arg Leu Cys Lys Leu Leu Gly His 225 230 235 240 cct gtc gat aac
gag aca gaa gct cta aaa atc ctt cgt caa gcc ccc 768 Pro Val Asp Asn
Glu Thr Glu Ala Leu Lys Ile Leu Arg Gln Ala Pro 245 250 255 ata gat
gat ctt ata gac aac aga ata aaa cca aaa gac aaa ggc caa 816 Ile Asp
Asp Leu Ile Asp Asn Arg Ile Lys Pro Lys Asp Lys Gly Gln 260 265 270
ctt att ata gac tat cct ttt cta cca aca ata gaa aaa cgt tat caa 864
Leu Ile Ile Asp Tyr Pro Phe Leu Pro Thr Ile Glu Lys Arg Tyr Gln 275
280 285 aat ttt gaa cca ttc ttg gac cag tct cca tta tca aaa atg caa
tca 912 Asn Phe Glu Pro Phe Leu Asp Gln Ser Pro Leu Ser Lys Met Gln
Ser 290 295 300 ggc aat ttc aca aaa gtc cca ttt ata tgt gga tac aac
agt gct gaa 960 Gly Asn Phe Thr Lys Val Pro Phe Ile Cys Gly Tyr Asn
Ser Ala Glu 305 310 315 320 gga att tta ggt tta atg gac ttc aag gat
gac cca aat ata ttt gag 1008 Gly Ile Leu Gly Leu Met Asp Phe Lys
Asp Asp Pro Asn Ile Phe Glu 325 330 335 aag ttt gaa gct gat ttt gaa
aga ttt gta cca gta gat ttg aat cta 1056 Lys Phe Glu Ala Asp Phe
Glu Arg Phe Val Pro Val Asp Leu Asn Leu 340 345 350 act tta agg tct
aag gaa tct aaa aaa ttg gct gaa gaa atg aga aag 1104 Thr Leu Arg
Ser Lys Glu Ser Lys Lys Leu Ala Glu Glu Met Arg Lys 355 360 365 ttt
tat tac caa gac gaa cct gtt tct tca gac aac aaa gaa aaa ttt 1152
Phe Tyr Tyr Gln Asp Glu Pro Val Ser Ser Asp Asn Lys Glu Lys Phe 370
375 380 gtc agt gtt att agt gat act tgg ttt ttg aga ggg att aaa aat
act 1200 Val Ser Val Ile Ser Asp Thr Trp Phe Leu Arg Gly Ile Lys
Asn Thr 385 390 395 400 gca aga tat ata att gaa cat tcc tca gaa ccg
tta tat tta tat gtt 1248 Ala Arg Tyr Ile Ile Glu His Ser Ser Glu
Pro Leu Tyr Leu Tyr Val 405 410 415 tat agt ttt gat gat ttt ggt ttt
ttg aag aaa ctt gta tta gat cct 1296 Tyr Ser Phe Asp Asp Phe Gly
Phe Leu Lys Lys Leu Val Leu Asp Pro 420 425 430 aat att gaa gga gca
gct cat gga gat gag ctg gga tat ctt ttc aag 1344 Asn Ile Glu Gly
Ala Ala His Gly Asp Glu Leu Gly Tyr Leu Phe Lys 435 440 445 atg agt
ttt aca gaa ttt cca aaa gat tta cca agt gca gtg gtg aat 1392 Met
Ser Phe Thr Glu Phe Pro Lys Asp Leu Pro Ser Ala Val Val Asn 450 455
460 agg gaa cga ttg ttg caa ctt tgg aca aat ttt gca aaa aca gga aat
1440 Arg Glu Arg Leu Leu Gln Leu Trp Thr Asn Phe Ala Lys Thr Gly
Asn 465 470 475 480 ccc act cct gaa atc aat gat gtt ata aca aca aaa
tgg gat aaa gct 1488 Pro Thr Pro Glu Ile Asn Asp Val Ile Thr Thr
Lys Trp Asp Lys Ala 485 490 495 act gag gaa aaa tca gat cat atg gat
atc gat aat act ttg aga atg 1536 Thr Glu Glu Lys Ser Asp His Met
Asp Ile Asp Asn Thr Leu Arg Met 500 505 510 att cca gat cct gat gca
aaa cga ctt aga ttt tgg aat aaa ttt tta 1584 Ile Pro Asp Pro Asp
Ala Lys Arg Leu Arg Phe Trp Asn Lys Phe Leu 515 520 525 52 1584 DNA
Ctenocephalides felis 52 taaaaattta ttccaaaatc taagtcgttt
tgcatcagga tctggaatca ttctcaaagt 60 attatcgata tccatatgat
ctgatttttc ctcagtagct ttatcccatt ttgttgttat 120 aacatcattg
atttcaggag tgggatttcc tgtttttgca aaatttgtcc aaagttgcaa 180
caatcgttcc ctattcacca ctgcacttgg taaatctttt ggaaattctg taaaactcat
240 cttgaaaaga tatcccagct catctccatg agctgctcct tcaatattag
gatctaatac 300 aagtttcttc aaaaaaccaa aatcatcaaa actataaaca
tataaatata acggttctga 360 ggaatgttca attatatatc ttgcagtatt
tttaatccct ctcaaaaacc aagtatcact 420 aataacactg acaaattttt
ctttgttgtc tgaagaaaca ggttcgtctt ggtaataaaa 480 ctttctcatt
tcttcagcca attttttaga ttccttagac cttaaagtta gattcaaatc 540
tactggtaca aatctttcaa aatcagcttc aaacttctca aatatatttg ggtcatcctt
600 gaagtccatt aaacctaaaa ttccttcagc actgttgtat ccacatataa
atgggacttt 660 tgtgaaattg cctgattgca tttttgataa tggagactgg
tccaagaatg gttcaaaatt 720 ttgataacgt ttttctattg ttggtagaaa
aggatagtct ataataagtt ggcctttgtc 780 ttttggtttt attctgttgt
ctataagatc atctatgggg gcttgacgaa ggatttttag 840 agcttctgtc
tcgttatcga caggatgacc cagaagtttg cagaggcgga atgcattttc 900
tttattatta tcatgttgaa gtgcccaagg attaaaagca cttccacttt gtgagatcgc
960 tttatgaaaa agtcctttgg aaagatctga taacatcaaa taatgaacac
ttgcaccacc 1020 tgctgattct ccaaaaatag tcacattgtt ggggtcacca
ccaaaggatg caatattgtt 1080 ttttacccat tttagagctt caacctggtc
catcaaacca acattgccag gcgcttcttc 1140 tattcccagg ttcaaaaatc
ccaaaacacc taatcgataa ttgaaagtaa ccagaacaat 1200 tccataatcc
atcaaatatt caggaccata catatcacta tttccagatc ccatgaagaa 1260
gcctcctcca tgtatccata ccattactgg aagaagtgat ttctctgatg tttttggtac
1320 atagacattg aggtataaac aatcttcagc ccctacttta atttttttaa
taaaatgtac 1380 tgatctacaa ctattccctt ctttactagc atcaagaaca
cctgaccaag gttctgcagg 1440 ttgaggtggc ttaaatctta gatcacctac
aggaggtttg gcatatggaa ttccagaata 1500 actatggaac acatttcctt
tttcactaat ttgctctttt ccttttaaag taccttgaag 1560 caaagtcact
tgtagatcag ccat 1584 53 530 PRT Ctenocephalides felis 53 Asp Pro
Pro Thr Val Thr Leu Pro Gln Gly Glu Leu Val Gly Lys Ala 1 5 10 15
Leu Thr Asn Glu Asn Gly Lys Glu Tyr Phe Ser Tyr Thr Gly Val Pro 20
25 30 Tyr Ala Lys Pro Pro Val Gly Glu Leu Arg Phe Lys Pro Pro Gln
Lys 35 40 45 Ala Glu Pro Trp Asn Gly Val Phe Asn Ala Thr Ser His
Gly Asn Val 50 55 60 Cys Lys Ala Leu Asn Phe Phe Leu Lys Lys Ile
Glu Gly Asp Glu Asp 65 70 75 80 Cys Leu Leu Val Asn Val Tyr Ala Pro
Lys Thr Thr Ser Asp Lys Lys 85 90 95 Leu Pro Val Phe Phe Trp Val
His Gly Gly Gly Phe Val Thr Gly Ser 100 105 110 Gly Asn Leu Glu Phe
Gln Ser Pro Asp Tyr Leu Val Asn Tyr Asp Val 115 120 125 Ile Phe Val
Thr Phe Asn Tyr Arg Leu Gly Pro Leu Gly Phe Leu Asn 130 135 140 Leu
Glu Leu Glu Gly Ala Pro Gly Asn Val Gly Leu Leu Asp Gln Val 145 150
155 160 Ala Ala Leu Lys Trp Thr Lys Glu Asn Ile Glu Lys Phe Gly Gly
Asp 165 170 175 Pro Glu Asn Ile Thr Ile Gly Gly Val Ser Ala Gly Gly
Ala Ser Val 180 185 190 His Tyr Leu Leu Leu Ser His Thr Thr Thr Gly
Leu Tyr Lys Arg Ala 195 200 205 Ile Ala Gln Ser Gly Ser Ala Leu Asn
Pro Trp Ala Phe Gln Arg His 210 215 220 Pro Val Lys Arg Ser Leu Gln
Leu Ala Glu Ile Leu Gly His Pro Thr 225 230 235 240 Asn Asn Thr Gln
Asp Ala Leu Glu Phe Leu Gln Lys Ala Pro Val Asp 245 250 255 Ser Leu
Leu Lys Lys Met Pro Ala Glu Thr Glu Gly Glu Ile Ile Glu 260 265 270
Glu Phe Val Phe Val Pro Ser Ile Glu Lys Val Phe Pro Ser His Gln 275
280 285 Pro Phe Leu Glu Glu Ser Pro Leu Ala Arg Met Lys Ser Gly Ser
Phe 290 295 300 Asn Lys Val Pro Leu Leu Val Gly Phe Asn Ser Ala Glu
Gly Leu Leu 305 310 315 320 Tyr Lys Phe Phe Met Lys Glu Lys Pro Glu
Met Leu Asn Gln Ala Glu 325 330 335 Ala Asp Phe Glu Arg Leu Val Pro
Ala Glu Phe Glu Leu Ala His Gly 340 345 350 Ser Glu Glu Ser Lys Lys
Leu Ala Glu Lys Ile Arg Lys Phe Tyr Phe 355 360 365 Asp Asp Lys Pro
Val Pro Glu Asn Glu Gln Lys Phe Ile Asp Leu Ile 370 375 380 Gly Asp
Ile Trp Phe Thr Arg Gly Ile Asp Lys His Val Lys Leu Ser 385 390 395
400 Val Glu Lys Gln Asp Glu Pro Val Tyr Tyr Tyr Glu Tyr Ser Phe Ser
405 410 415 Glu Ser His Pro Ala Lys Gly Thr Phe Gly Asp His Asn Leu
Thr Gly 420 425 430 Ala Cys His Gly Glu Glu Leu Val Asn Leu Phe Lys
Val Glu Met Met 435 440 445 Lys Leu Glu Lys Asp Lys Pro Asn Val Leu
Leu Thr Lys Asp Arg Val 450 455 460 Leu Ala Met Trp Thr Asn Phe Ile
Lys Asn Gly Asn Pro Thr Pro Glu 465 470 475 480 Val Thr Glu Leu Leu
Pro Val Lys Trp Glu Pro Ala Thr Lys Asp Lys 485 490 495 Leu Asn Tyr
Leu Asn Ile Asp Ala Thr Leu Thr Leu Gly Thr Asn Pro 500 505 510 Glu
Glu Thr Arg Val Lys Phe Trp Glu Asp Ala Thr Lys Thr Leu His 515 520
525 Ser Gln 530 54 570 PRT Ctenocephalides felis 54 Trp Asp Asn Leu
Asp Gln His Leu Cys Arg Val Gln Phe Asn Gly Ile 1 5 10 15 Thr Glu
Gly Lys Pro Phe Arg Tyr Lys Asp His Arg Asn Asp Val Tyr 20 25 30
Cys Ser Tyr Leu Gly Ile Pro Tyr Ala Glu Pro Pro Phe Gly Pro Leu 35
40 45 Arg Phe Gln Ser Pro Lys Pro Ile Ser Asn Pro Lys Thr Gly Phe
Val 50 55 60 Gln Ala Arg Thr Leu Gly Asp Lys Cys Phe Gln Glu Ser
Leu Ile Tyr 65 70 75 80 Ser Tyr Ala Gly Ser Glu Asp Cys Leu Tyr Leu
Asn Ile Phe Thr Pro 85 90 95 Glu Thr Val Asn Ser Ala Asn Asn Thr
Lys Tyr Pro Val Met Phe Trp 100 105 110 Ile His Gly Gly Ala Phe Asn
Gln Gly Ser Gly Ser Tyr Asn Phe Phe 115 120 125 Gly Pro Asp Tyr Leu
Ile Arg Glu Gly Ile Ile Leu Val Thr Ile Asn 130 135 140 Tyr Arg Leu
Gly Val Phe Gly Phe Leu Ser Ala Pro Glu Trp Asp Ile 145 150 155 160
His Gly Asn Met Gly Leu Lys Asp Gln Arg Leu Ala Leu Lys Trp Val 165
170 175 Tyr Asp Asn Ile Glu Lys Phe Gly Gly Asp Arg Glu Lys Ile Thr
Ile 180 185 190 Ala Gly Glu Ser Ala Gly Ala Ala Ser Val His Phe Leu
Met Met Asp 195 200 205 Asn Ser Thr Arg Lys Tyr Tyr Gln Arg Ala Ile
Leu Gln Ser Gly Thr 210 215 220 Leu Leu Asn Pro Thr Ala Asn Gln Ile
Gln Leu Leu His Arg Phe Glu 225 230 235 240 Lys Leu Lys Gln Val Leu
Asn Ile Thr Gln Lys Gln Glu Leu Leu Asn 245 250 255 Leu Asp Lys Asn
Leu Ile Leu Arg Ala Ala Leu Asn Arg Val Pro Asp 260 265 270 Ser Asn
Asp His Asp Arg Asp Thr Val Pro Val Phe Asn Pro Val Leu 275 280 285
Glu Ser Pro Glu Ser Pro Asp Pro Ile Thr Phe Pro Ser Ala Leu Glu 290
295 300 Arg Met Arg Asn Gly Glu Phe Pro Asp Val Asp Val Ile Ile Gly
Phe 305 310 315 320 Asn Ser Ala Glu Gly Leu Arg Ser Met Ala Arg Val
Thr Arg Gly Asn 325 330 335 Met Glu Val His Lys Thr Leu Thr Asn Ile
Glu Arg Ala Ile Pro Arg 340 345 350 Asp Ala Asn Ile Trp Lys Asn Pro
Asn Gly Ile Glu Glu Lys Lys Leu 355 360 365 Ile Lys Met Leu Thr Glu
Phe Tyr Asp Gln Val Lys Glu Gln Asn Asp 370 375 380 Asp Ile Glu Ala
Tyr Val Gln Leu Lys Gly Asp Ala Gly Tyr Leu Gln 385 390 395 400 Gly
Ile Tyr Arg Thr Leu Lys Ala Ile Phe Phe Asn Glu Phe Arg Arg 405 410
415 Asn Ser Asn Leu Tyr Leu Tyr Arg Leu Ser Asp Asp Thr Tyr Ser Val
420 425 430 Tyr Lys Ser Tyr Ile Leu Pro Tyr Arg Trp Gly Ser Leu Pro
Gly Val 435 440 445 Ser His Gly Asp Asp Leu Gly Tyr Leu Phe Ala Asn
Ser Leu Asp Val 450 455 460 Pro Ile Leu Gly Thr Thr His Ile Ser Ile
Pro Gln Asp Ala Met Gln 465 470 475 480 Thr Leu Glu Arg Met Val Arg
Ile Trp Thr Asn Phe Val Lys Asn Gly 485 490 495 Lys Pro Thr Ser Asn
Thr Glu Asp Ala Ser Cys Asp Thr Lys Arg His 500 505 510 Leu Asn Asp
Ile Phe Trp Glu Pro Tyr Asn Asp Glu Glu Pro Lys Tyr 515 520 525 Leu
Asp Met Gly Lys Glu Asn Phe Glu Met Lys Asn Ile Leu Glu Leu 530 535
540 Lys Arg Met Met Leu Trp Asp Glu Val Tyr Arg Asn Ala Asn Leu Arg
545 550 555 560 Phe Arg Val Cys Asn Glu Gly Ser Ile Arg 565 570 55
570 PRT Ctenocephalides felis 55 Trp Asp Asn Leu Asp Gln His Leu
Cys Arg Val Gln Phe Asn Gly Ile 1 5 10 15 Thr Glu Gly Lys Pro Phe
Arg Tyr Lys Asp His Lys Asn Asp Val Tyr 20 25 30 Cys Ser Tyr Leu
Gly Ile Pro Tyr Ala Glu Pro Pro Ile Gly Pro Leu 35 40 45 Arg Phe
Gln Ser Pro Lys Pro Ile Ser Asn Pro Lys Thr Gly Phe Val 50 55 60
Gln Ala Arg Ser Leu Gly Asp Lys Cys Phe Gln Glu Ser Leu Ile Tyr 65
70 75 80 Ser Tyr Ala Gly Ser Glu Asp Cys Leu Tyr Leu Asn Ile Phe
Thr Pro 85 90 95 Glu Thr Val Asn Ser Ala Asn Asn Thr Lys Tyr Pro
Val Met Phe Trp 100 105 110 Ile His Gly Gly Ala Phe Asn Gln Gly Ser
Gly Ser Tyr Asn Phe Phe 115 120 125 Gly Pro Asp Tyr Leu Ile Arg Glu
Gly Ile Ile Leu Val Thr Ile Asn 130 135 140 Tyr Arg Leu Gly Val Phe
Gly Phe Leu Ser Ala Pro Glu Trp Asp Ile 145 150 155 160 His Gly Asn
Met Gly Leu Lys Asp Gln Arg Leu Ala Leu Lys Trp Val 165 170 175 Tyr
Asp Asn Ile Glu Lys Phe Gly Gly Asp Arg Asp Lys Ile Thr Ile 180 185
190 Ala Gly Glu Ser Ala Gly Ala Ala Ser Val His Phe Leu Met Met Asp
195 200 205 Asn Ser Thr Arg Lys Tyr Tyr Gln Arg Ala Ile Leu Gln Ser
Gly Thr 210 215 220 Leu Leu Asn Pro Thr Ala Asn Gln Ile Gln Pro Leu
His Arg Phe Glu 225 230 235 240 Lys Leu Lys Gln Val Leu Asn Ile Thr
Gln Lys Gln Glu Leu Leu Asn 245 250 255 Leu Asp Lys Asn Gln Ile Leu
Arg Ala Ala Leu Asn Arg Val Pro Asp 260 265 270 Asn Asn Asp His Glu
Arg Asp Thr Val Pro Val Phe Asn Pro Val Leu 275 280 285 Glu Ser Pro
Glu Ser Pro Asp Pro Ile Thr Phe Pro Ser Ala Leu Glu 290 295 300 Arg
Met Arg Asn Gly Glu Phe Pro Asp Val Asp Val Ile Ile Gly Phe 305 310
315 320 Asn Ser Ala Glu Gly Leu Arg Ser Met Pro Arg Val Thr Arg Gly
Asn 325 330 335 Met Glu Val Tyr Lys Thr Leu Thr Asn Ile Glu Arg Ala
Ile Pro Arg 340 345 350 Asp Ala Asn Ile Trp Lys Asn Pro Asn Gly Ile
Glu Glu Lys Lys Leu 355 360 365 Ile Lys Met Leu Thr Glu Phe Tyr Asp
Gln Val Lys Glu Gln Asn Asp 370 375 380 Asp Ile Glu Ala Tyr Val Gln
Leu Lys Gly Asp Ala Gly Tyr Leu Gln 385 390 395 400 Gly Ile Tyr Arg
Thr Leu Lys Ala Ile Phe Phe Asn Glu Ile Lys Arg 405 410 415 Asn Ser
Asn Leu Tyr Leu Tyr Arg Leu Ser Asp Asp Thr Tyr Ser Val 420 425 430
Tyr Lys Ser Tyr Ile Leu Pro Tyr Arg Trp Gly Ser Leu Pro Gly Val 435
440 445 Ser His Gly Asp Asp Leu Gly Tyr Leu Phe Ala Asn Ser Leu Asp
Val 450 455 460 Pro Ile Leu Gly Thr Thr His Ile Ser Ile Pro Gln Asp
Ala Met Gln 465 470 475 480 Thr Leu Glu Arg Met Val Arg Ile Trp Thr
Asn Phe Val Lys Asn Gly 485 490 495 Lys Pro Thr Ser Asn Thr Glu Asp
Ala Ser Cys Asp Thr Lys Arg His 500 505 510 Leu Asn Asp Ile Phe Trp
Glu Pro Tyr Asn Asp Glu Glu Pro Lys Tyr 515 520 525 Leu Asp Met Gly
Lys Glu His
Phe Glu Met Lys Asn Ile Leu Glu Leu 530 535 540 Lys Arg Met Met Leu
Trp Asp Glu Val Tyr Arg Asn Ala Asn Leu Arg 545 550 555 560 Phe Arg
Val Cys Asn Glu Gly Ser Ile Arg 565 570 56 20 DNA Artificial
sequence Synthetic Primer 56 gtgcgtacac gtttactacc 20 57 2144 DNA
Ctenocephalides felis CDS (30)..(1682) 57 gtacacatag tcaatagtct
agatccaag atg tct cgt gtt att ttt tta agt 53 Met Ser Arg Val Ile
Phe Leu Ser 1 5 tgt att ttt ttg ttt agt ttt aat ttt ata aaa tgt gat
tcc ccg act 101 Cys Ile Phe Leu Phe Ser Phe Asn Phe Ile Lys Cys Asp
Ser Pro Thr 10 15 20 gta act ttg ccc caa ggc gaa ttg gtt gga aaa
gct ttg acg aac gaa 149 Val Thr Leu Pro Gln Gly Glu Leu Val Gly Lys
Ala Leu Thr Asn Glu 25 30 35 40 aat gga aaa gag tat ttt agc tac aca
ggt gta cct tat gct aaa cct 197 Asn Gly Lys Glu Tyr Phe Ser Tyr Thr
Gly Val Pro Tyr Ala Lys Pro 45 50 55 cct gtt gga gaa ctt aga ttt
aag cct cca cag aaa gct gag cca tgg 245 Pro Val Gly Glu Leu Arg Phe
Lys Pro Pro Gln Lys Ala Glu Pro Trp 60 65 70 caa ggt gtt ttc aac
gcc aca tta tac gga aat gtg tgt aaa tct tta 293 Gln Gly Val Phe Asn
Ala Thr Leu Tyr Gly Asn Val Cys Lys Ser Leu 75 80 85 aat ttc ttc
ttg aag aaa att gaa gga gac gaa gac tgc ttg gta gta 341 Asn Phe Phe
Leu Lys Lys Ile Glu Gly Asp Glu Asp Cys Leu Val Val 90 95 100 aac
gtg tac gca cca aaa aca act tct gat aaa aaa ctt cca gta ttt 389 Asn
Val Tyr Ala Pro Lys Thr Thr Ser Asp Lys Lys Leu Pro Val Phe 105 110
115 120 ttc tgg gtt cat ggt ggt ggt ttt gtg act gga tcc gga aat tta
gaa 437 Phe Trp Val His Gly Gly Gly Phe Val Thr Gly Ser Gly Asn Leu
Glu 125 130 135 ttc caa agc cca gat tat tta gta rat ttt gat gtt att
ttc gta act 485 Phe Gln Ser Pro Asp Tyr Leu Val Xaa Phe Asp Val Ile
Phe Val Thr 140 145 150 ttc aat tac cga ttg gga cct ctc gga ttt ctg
aat ttg gag ttg gag 533 Phe Asn Tyr Arg Leu Gly Pro Leu Gly Phe Leu
Asn Leu Glu Leu Glu 155 160 165 ggt gct cca gga aat gta gga tta ttg
gat cag gtg gca gct ctg aaa 581 Gly Ala Pro Gly Asn Val Gly Leu Leu
Asp Gln Val Ala Ala Leu Lys 170 175 180 tgg acc aaa gaa aac att gag
aaa ttt ggt gga gat cca gaa aat att 629 Trp Thr Lys Glu Asn Ile Glu
Lys Phe Gly Gly Asp Pro Glu Asn Ile 185 190 195 200 aca att ggt ggt
gtt tct gct ggt gga gca agt gtt cat tat ctt ttg 677 Thr Ile Gly Gly
Val Ser Ala Gly Gly Ala Ser Val His Tyr Leu Leu 205 210 215 tta tct
cat aca acc act gga ctt tac aaa agg gca att gct caa agt 725 Leu Ser
His Thr Thr Thr Gly Leu Tyr Lys Arg Ala Ile Ala Gln Ser 220 225 230
gga agt gct ttt aat cca tgg gcc ttc caa aga cat cca gta aag cgt 773
Gly Ser Ala Phe Asn Pro Trp Ala Phe Gln Arg His Pro Val Lys Arg 235
240 245 agt ctt caa ctt gct gag ata ttg ggt cat ccc aca aac aat act
caa 821 Ser Leu Gln Leu Ala Glu Ile Leu Gly His Pro Thr Asn Asn Thr
Gln 250 255 260 gat gct tta gaa ttc tta caa aaa gcc ccc gta gac agt
ctc ctg aag 869 Asp Ala Leu Glu Phe Leu Gln Lys Ala Pro Val Asp Ser
Leu Leu Lys 265 270 275 280 aaa atg cca gct gaa aca gaa ggt gaa ata
ata gaa gag ttt gtc ttc 917 Lys Met Pro Ala Glu Thr Glu Gly Glu Ile
Ile Glu Glu Phe Val Phe 285 290 295 gta cca tca att gaa aaa gtt ttc
cca tcc cac caa cct ttc ttg gaa 965 Val Pro Ser Ile Glu Lys Val Phe
Pro Ser His Gln Pro Phe Leu Glu 300 305 310 gaa tca cca ttg gcc aga
atg aaa tcc gga tcc ttt aac aaa gta cct 1013 Glu Ser Pro Leu Ala
Arg Met Lys Ser Gly Ser Phe Asn Lys Val Pro 315 320 325 tta tta gtt
gga ttt aac agt gca gaa gga ctt ttg ttc aaa ttc ttc 1061 Leu Leu
Val Gly Phe Asn Ser Ala Glu Gly Leu Leu Phe Lys Phe Phe 330 335 340
atg aaa gaa aaa cca gag atg ctg aac caa gct gaa gca gat ttt gaa
1109 Met Lys Glu Lys Pro Glu Met Leu Asn Gln Ala Glu Ala Asp Phe
Glu 345 350 355 360 aga ctc gta cca gcc gaa ttt gaa tta gtc cat gga
tca gag gaa tcg 1157 Arg Leu Val Pro Ala Glu Phe Glu Leu Val His
Gly Ser Glu Glu Ser 365 370 375 aaa aaa ctt gca gaa aaa atc agg aag
ttt tac ttt gac gat aaa ccc 1205 Lys Lys Leu Ala Glu Lys Ile Arg
Lys Phe Tyr Phe Asp Asp Lys Pro 380 385 390 gtt cca gaa aat gaa cag
aaa ttt att gac ttg ata gga gat att tgg 1253 Val Pro Glu Asn Glu
Gln Lys Phe Ile Asp Leu Ile Gly Asp Ile Trp 395 400 405 ttt act aga
ggt gtt gac aag cat gtc aag ttg tct gtg gag aaa caa 1301 Phe Thr
Arg Gly Val Asp Lys His Val Lys Leu Ser Val Glu Lys Gln 410 415 420
gac gaa cca gtt tat tat tat gaa tat tcc ttc tcg gaa agt cat cct
1349 Asp Glu Pro Val Tyr Tyr Tyr Glu Tyr Ser Phe Ser Glu Ser His
Pro 425 430 435 440 gca aaa gga aca ttt ggt gat cat aat ctg act ggt
gca tgc cat gga 1397 Ala Lys Gly Thr Phe Gly Asp His Asn Leu Thr
Gly Ala Cys His Gly 445 450 455 gaa gaa ctt gtg aat tta ttc aaa gtc
gag atg atg aag ctg gaa aaa 1445 Glu Glu Leu Val Asn Leu Phe Lys
Val Glu Met Met Lys Leu Glu Lys 460 465 470 gat aaa cct aat gtt cta
tta aca aaa gat aga gta ctt gcc atg tgg 1493 Asp Lys Pro Asn Val
Leu Leu Thr Lys Asp Arg Val Leu Ala Met Trp 475 480 485 act aac ttc
atc aaa aat gga aat cct act cct gaa gta aca gaa tta 1541 Thr Asn
Phe Ile Lys Asn Gly Asn Pro Thr Pro Glu Val Thr Glu Leu 490 495 500
ttg cca gtt aaa tgg gaa cct gcc aca aaa gac aag ttg aat tat ttg
1589 Leu Pro Val Lys Trp Glu Pro Ala Thr Lys Asp Lys Leu Asn Tyr
Leu 505 510 515 520 aac att gat gcc acc tta act ttg gga aca aat cct
gag gca aac cga 1637 Asn Ile Asp Ala Thr Leu Thr Leu Gly Thr Asn
Pro Glu Ala Asn Arg 525 530 535 gtc aaa ttt tgg gaa gac gcc aca aaa
tct ttg cac ggt caa taa 1682 Val Lys Phe Trp Glu Asp Ala Thr Lys
Ser Leu His Gly Gln 540 545 550 taatttatga aaattgtttt aaatacttta
ggtaatatat taggtaaata aaaattaaaa 1742 aataacaatt tttatgtttt
atgtattggc ttatgtgtat cagttctaat tttatttatt 1802 tattcttgtt
ttgcttgttt tgaaatatca tggttttaat tttcaaaaca caacgtcgtt 1862
tgtttttagc aaaatttcca atagatatgt tatattaagt actctgaagt atttttatat
1922 atacactaaa atcagtaaaa atacattaac taaaaatata agatattttc
aataattttt 1982 tttaaagaaa ataccaaaaa taaagtaaaa ttccaaacgg
aatttttgtt taacttaaaa 2042 ataaaattaa ctcttcaata attttgataa
ttagtatttc tgatatcatt agtgaaaatt 2102 atattttgat aatacgtatt
tatatttaaa ataaaattat gt 2144 58 550 PRT Ctenocephalides felis
misc_feature (145)..(145) The 'Xaa' at location 145 stands for Asp,
or Asn. 58 Met Ser Arg Val Ile Phe Leu Ser Cys Ile Phe Leu Phe Ser
Phe Asn 1 5 10 15 Phe Ile Lys Cys Asp Ser Pro Thr Val Thr Leu Pro
Gln Gly Glu Leu 20 25 30 Val Gly Lys Ala Leu Thr Asn Glu Asn Gly
Lys Glu Tyr Phe Ser Tyr 35 40 45 Thr Gly Val Pro Tyr Ala Lys Pro
Pro Val Gly Glu Leu Arg Phe Lys 50 55 60 Pro Pro Gln Lys Ala Glu
Pro Trp Gln Gly Val Phe Asn Ala Thr Leu 65 70 75 80 Tyr Gly Asn Val
Cys Lys Ser Leu Asn Phe Phe Leu Lys Lys Ile Glu 85 90 95 Gly Asp
Glu Asp Cys Leu Val Val Asn Val Tyr Ala Pro Lys Thr Thr 100 105 110
Ser Asp Lys Lys Leu Pro Val Phe Phe Trp Val His Gly Gly Gly Phe 115
120 125 Val Thr Gly Ser Gly Asn Leu Glu Phe Gln Ser Pro Asp Tyr Leu
Val 130 135 140 Xaa Phe Asp Val Ile Phe Val Thr Phe Asn Tyr Arg Leu
Gly Pro Leu 145 150 155 160 Gly Phe Leu Asn Leu Glu Leu Glu Gly Ala
Pro Gly Asn Val Gly Leu 165 170 175 Leu Asp Gln Val Ala Ala Leu Lys
Trp Thr Lys Glu Asn Ile Glu Lys 180 185 190 Phe Gly Gly Asp Pro Glu
Asn Ile Thr Ile Gly Gly Val Ser Ala Gly 195 200 205 Gly Ala Ser Val
His Tyr Leu Leu Leu Ser His Thr Thr Thr Gly Leu 210 215 220 Tyr Lys
Arg Ala Ile Ala Gln Ser Gly Ser Ala Phe Asn Pro Trp Ala 225 230 235
240 Phe Gln Arg His Pro Val Lys Arg Ser Leu Gln Leu Ala Glu Ile Leu
245 250 255 Gly His Pro Thr Asn Asn Thr Gln Asp Ala Leu Glu Phe Leu
Gln Lys 260 265 270 Ala Pro Val Asp Ser Leu Leu Lys Lys Met Pro Ala
Glu Thr Glu Gly 275 280 285 Glu Ile Ile Glu Glu Phe Val Phe Val Pro
Ser Ile Glu Lys Val Phe 290 295 300 Pro Ser His Gln Pro Phe Leu Glu
Glu Ser Pro Leu Ala Arg Met Lys 305 310 315 320 Ser Gly Ser Phe Asn
Lys Val Pro Leu Leu Val Gly Phe Asn Ser Ala 325 330 335 Glu Gly Leu
Leu Phe Lys Phe Phe Met Lys Glu Lys Pro Glu Met Leu 340 345 350 Asn
Gln Ala Glu Ala Asp Phe Glu Arg Leu Val Pro Ala Glu Phe Glu 355 360
365 Leu Val His Gly Ser Glu Glu Ser Lys Lys Leu Ala Glu Lys Ile Arg
370 375 380 Lys Phe Tyr Phe Asp Asp Lys Pro Val Pro Glu Asn Glu Gln
Lys Phe 385 390 395 400 Ile Asp Leu Ile Gly Asp Ile Trp Phe Thr Arg
Gly Val Asp Lys His 405 410 415 Val Lys Leu Ser Val Glu Lys Gln Asp
Glu Pro Val Tyr Tyr Tyr Glu 420 425 430 Tyr Ser Phe Ser Glu Ser His
Pro Ala Lys Gly Thr Phe Gly Asp His 435 440 445 Asn Leu Thr Gly Ala
Cys His Gly Glu Glu Leu Val Asn Leu Phe Lys 450 455 460 Val Glu Met
Met Lys Leu Glu Lys Asp Lys Pro Asn Val Leu Leu Thr 465 470 475 480
Lys Asp Arg Val Leu Ala Met Trp Thr Asn Phe Ile Lys Asn Gly Asn 485
490 495 Pro Thr Pro Glu Val Thr Glu Leu Leu Pro Val Lys Trp Glu Pro
Ala 500 505 510 Thr Lys Asp Lys Leu Asn Tyr Leu Asn Ile Asp Ala Thr
Leu Thr Leu 515 520 525 Gly Thr Asn Pro Glu Ala Asn Arg Val Lys Phe
Trp Glu Asp Ala Thr 530 535 540 Lys Ser Leu His Gly Gln 545 550 59
2144 DNA Ctenocephalides felis 59 acataatttt attttaaata taaatacgta
ttatcaaaat ataattttca ctaatgatat 60 cagaaatact aattatcaaa
attattgaag agttaatttt atttttaagt taaacaaaaa 120 ttccgtttgg
aattttactt tatttttggt attttcttta aaaaaaatta ttgaaaatat 180
cttatatttt tagttaatgt atttttactg attttagtgt atatataaaa atacttcaga
240 gtacttaata taacatatct attggaaatt ttgctaaaaa caaacgacgt
tgtgttttga 300 aaattaaaac catgatattt caaaacaagc aaaacaagaa
taaataaata aaattagaac 360 tgatacacat aagccaatac ataaaacata
aaaattgtta ttttttaatt tttatttacc 420 taatatatta cctaaagtat
ttaaaacaat tttcataaat tattattgac cgtgcaaaga 480 ttttgtggcg
tcttcccaaa atttgactcg gtttgcctca ggatttgttc ccaaagttaa 540
ggtggcatca atgttcaaat aattcaactt gtcttttgtg gcaggttccc atttaactgg
600 caataattct gttacttcag gagtaggatt tccatttttg atgaagttag
tccacatggc 660 aagtactcta tcttttgtta atagaacatt aggtttatct
ttttccagct tcatcatctc 720 gactttgaat aaattcacaa gttcttctcc
atggcatgca ccagtcagat tatgatcacc 780 aaatgttcct tttgcaggat
gactttccga gaaggaatat tcataataat aaactggttc 840 gtcttgtttc
tccacagaca acttgacatg cttgtcaaca cctctagtaa accaaatatc 900
tcctatcaag tcaataaatt tctgttcatt ttctggaacg ggtttatcgt caaagtaaaa
960 cttcctgatt ttttctgcaa gttttttcga ttcctctgat ccatggacta
attcaaattc 1020 ggctggtacg agtctttcaa aatctgcttc agcttggttc
agcatctctg gtttttcttt 1080 catgaagaat ttgaacaaaa gtccttctgc
actgttaaat ccaactaata aaggtacttt 1140 gttaaaggat ccggatttca
ttctggccaa tggtgattct tccaagaaag gttggtggga 1200 tgggaaaact
ttttcaattg atggtacgaa gacaaactct tctattattt caccttctgt 1260
ttcagctggc attttcttca ggagactgtc tacgggggct ttttgtaaga attctaaagc
1320 atcttgagta ttgtttgtgg gatgacccaa tatctcagca agttgaagac
tacgctttac 1380 tggatgtctt tggaaggccc atggattaaa agcacttcca
ctttgagcaa ttgccctttt 1440 gtaaagtcca gtggttgtat gagataacaa
aagataatga acacttgctc caccagcaga 1500 aacaccacca attgtaatat
tttctggatc tccaccaaat ttctcaatgt tttctttggt 1560 ccatttcaga
gctgccacct gatccaataa tcctacattt cctggagcac cctccaactc 1620
caaattcaga aatccgagag gtcccaatcg gtaattgaaa gttacgaaaa taacatcaaa
1680 atytactaaa taatctgggc tttggaattc taaatttccg gatccagtca
caaaaccacc 1740 accatgaacc cagaaaaata ctggaagttt tttatcagaa
gttgtttttg gtgcgtacac 1800 gtttactacc aagcagtctt cgtctccttc
aattttcttc aagaagaaat ttaaagattt 1860 acacacattt ccgtataatg
tggcgttgaa aacaccttgc catggctcag ctttctgtgg 1920 aggcttaaat
ctaagttctc caacaggagg tttagcataa ggtacacctg tgtagctaaa 1980
atactctttt ccattttcgt tcgtcaaagc ttttccaacc aattcgcctt ggggcaaagt
2040 tacagtcggg gaatcacatt ttataaaatt aaaactaaac aaaaaaatac
aacttaaaaa 2100 aataacacga gacatcttgg atctagacta ttgactatgt gtac
2144 60 1650 DNA Ctenocephalides felis exon (1)..(1650) 60 atg tct
cgt gtt att ttt tta agt tgt att ttt ttg ttt agt ttt aat 48 Met Ser
Arg Val Ile Phe Leu Ser Cys Ile Phe Leu Phe Ser Phe Asn 1 5 10 15
ttt ata aaa tgt gat tcc ccg act gta act ttg ccc caa ggc gaa ttg 96
Phe Ile Lys Cys Asp Ser Pro Thr Val Thr Leu Pro Gln Gly Glu Leu 20
25 30 gtt gga aaa gct ttg acg aac gaa aat gga aaa gag tat ttt agc
tac 144 Val Gly Lys Ala Leu Thr Asn Glu Asn Gly Lys Glu Tyr Phe Ser
Tyr 35 40 45 aca ggt gta cct tat gct aaa cct cct gtt gga gaa ctt
aga ttt aag 192 Thr Gly Val Pro Tyr Ala Lys Pro Pro Val Gly Glu Leu
Arg Phe Lys 50 55 60 cct cca cag aaa gct gag cca tgg caa ggt gtt
ttc aac gcc aca tta 240 Pro Pro Gln Lys Ala Glu Pro Trp Gln Gly Val
Phe Asn Ala Thr Leu 65 70 75 80 tac gga aat gtg tgt aaa tct tta aat
ttc ttc ttg aag aaa att gaa 288 Tyr Gly Asn Val Cys Lys Ser Leu Asn
Phe Phe Leu Lys Lys Ile Glu 85 90 95 gga gac gaa gac tgc ttg gta
gta aac gtg tac gca cca aaa aca act 336 Gly Asp Glu Asp Cys Leu Val
Val Asn Val Tyr Ala Pro Lys Thr Thr 100 105 110 tct gat aaa aaa ctt
cca gta ttt ttc tgg gtt cat ggt ggt ggt ttt 384 Ser Asp Lys Lys Leu
Pro Val Phe Phe Trp Val His Gly Gly Gly Phe 115 120 125 gtg act gga
tcc gga aat tta gaa ttc caa agc cca gat tat tta gta 432 Val Thr Gly
Ser Gly Asn Leu Glu Phe Gln Ser Pro Asp Tyr Leu Val 130 135 140 rat
ttt gat gtt att ttc gta act ttc aat tac cga ttg gga cct ctc 480 Xaa
Phe Asp Val Ile Phe Val Thr Phe Asn Tyr Arg Leu Gly Pro Leu 145 150
155 160 gga ttt ctg aat ttg gag ttg gag ggt gct cca gga aat gta gga
tta 528 Gly Phe Leu Asn Leu Glu Leu Glu Gly Ala Pro Gly Asn Val Gly
Leu 165 170 175 ttg gat cag gtg gca gct ctg aaa tgg acc aaa gaa aac
att gag aaa 576 Leu Asp Gln Val Ala Ala Leu Lys Trp Thr Lys Glu Asn
Ile Glu Lys 180 185 190 ttt ggt gga gat cca gaa aat att aca att ggt
ggt gtt tct gct ggt 624 Phe Gly Gly Asp Pro Glu Asn Ile Thr Ile Gly
Gly Val Ser Ala Gly 195 200 205 gga gca agt gtt cat tat ctt ttg tta
tct cat aca acc act gga ctt 672 Gly Ala Ser Val His Tyr Leu Leu Leu
Ser His Thr Thr Thr Gly Leu 210 215 220 tac aaa agg gca att gct caa
agt gga agt gct ttt aat cca tgg gcc 720 Tyr Lys Arg Ala Ile Ala Gln
Ser Gly Ser Ala Phe Asn Pro Trp Ala 225 230 235 240 ttc caa aga cat
cca gta aag cgt agt ctt caa ctt gct gag ata ttg 768 Phe Gln Arg His
Pro Val Lys Arg Ser Leu Gln Leu Ala Glu Ile Leu 245 250 255 ggt cat
ccc aca aac aat act caa gat gct tta gaa ttc tta caa aaa 816 Gly His
Pro Thr Asn Asn Thr Gln Asp Ala Leu Glu Phe Leu Gln Lys 260 265 270
gcc ccc gta gac agt ctc ctg aag aaa atg cca gct gaa aca gaa ggt 864
Ala Pro Val Asp Ser Leu Leu Lys Lys Met Pro Ala Glu Thr Glu Gly 275
280 285 gaa ata ata gaa gag ttt gtc ttc gta cca tca att gaa aaa gtt
ttc 912 Glu Ile Ile
Glu Glu Phe Val Phe Val Pro Ser Ile Glu Lys Val Phe 290 295 300 cca
tcc cac caa cct ttc ttg gaa gaa tca cca ttg gcc aga atg aaa 960 Pro
Ser His Gln Pro Phe Leu Glu Glu Ser Pro Leu Ala Arg Met Lys 305 310
315 320 tcc gga tcc ttt aac aaa gta cct tta tta gtt gga ttt aac agt
gca 1008 Ser Gly Ser Phe Asn Lys Val Pro Leu Leu Val Gly Phe Asn
Ser Ala 325 330 335 gaa gga ctt ttg ttc aaa ttc ttc atg aaa gaa aaa
cca gag atg ctg 1056 Glu Gly Leu Leu Phe Lys Phe Phe Met Lys Glu
Lys Pro Glu Met Leu 340 345 350 aac caa gct gaa gca gat ttt gaa aga
ctc gta cca gcc gaa ttt gaa 1104 Asn Gln Ala Glu Ala Asp Phe Glu
Arg Leu Val Pro Ala Glu Phe Glu 355 360 365 tta gtc cat gga tca gag
gaa tcg aaa aaa ctt gca gaa aaa atc agg 1152 Leu Val His Gly Ser
Glu Glu Ser Lys Lys Leu Ala Glu Lys Ile Arg 370 375 380 aag ttt tac
ttt gac gat aaa ccc gtt cca gaa aat gaa cag aaa ttt 1200 Lys Phe
Tyr Phe Asp Asp Lys Pro Val Pro Glu Asn Glu Gln Lys Phe 385 390 395
400 att gac ttg ata gga gat att tgg ttt act aga ggt gtt gac aag cat
1248 Ile Asp Leu Ile Gly Asp Ile Trp Phe Thr Arg Gly Val Asp Lys
His 405 410 415 gtc aag ttg tct gtg gag aaa caa gac gaa cca gtt tat
tat tat gaa 1296 Val Lys Leu Ser Val Glu Lys Gln Asp Glu Pro Val
Tyr Tyr Tyr Glu 420 425 430 tat tcc ttc tcg gaa agt cat cct gca aaa
gga aca ttt ggt gat cat 1344 Tyr Ser Phe Ser Glu Ser His Pro Ala
Lys Gly Thr Phe Gly Asp His 435 440 445 aat ctg act ggt gca tgc cat
gga gaa gaa ctt gtg aat tta ttc aaa 1392 Asn Leu Thr Gly Ala Cys
His Gly Glu Glu Leu Val Asn Leu Phe Lys 450 455 460 gtc gag atg atg
aag ctg gaa aaa gat aaa cct aat gtt cta tta aca 1440 Val Glu Met
Met Lys Leu Glu Lys Asp Lys Pro Asn Val Leu Leu Thr 465 470 475 480
aaa gat aga gta ctt gcc atg tgg act aac ttc atc aaa aat gga aat
1488 Lys Asp Arg Val Leu Ala Met Trp Thr Asn Phe Ile Lys Asn Gly
Asn 485 490 495 cct act cct gaa gta aca gaa tta ttg cca gtt aaa tgg
gaa cct gcc 1536 Pro Thr Pro Glu Val Thr Glu Leu Leu Pro Val Lys
Trp Glu Pro Ala 500 505 510 aca aaa gac aag ttg aat tat ttg aac att
gat gcc acc tta act ttg 1584 Thr Lys Asp Lys Leu Asn Tyr Leu Asn
Ile Asp Ala Thr Leu Thr Leu 515 520 525 gga aca aat cct gag gca aac
cga gtc aaa ttt tgg gaa gac gcc aca 1632 Gly Thr Asn Pro Glu Ala
Asn Arg Val Lys Phe Trp Glu Asp Ala Thr 530 535 540 aaa tct ttg cac
ggt caa 1650 Lys Ser Leu His Gly Gln 545 550 61 1650 DNA
Ctenocephalides felis 61 ttgaccgtgc aaagattttg tggcgtcttc
ccaaaatttg actcggtttg cctcaggatt 60 tgttcccaaa gttaaggtgg
catcaatgtt caaataattc aacttgtctt ttgtggcagg 120 ttcccattta
actggcaata attctgttac ttcaggagta ggatttccat ttttgatgaa 180
gttagtccac atggcaagta ctctatcttt tgttaataga acattaggtt tatctttttc
240 cagcttcatc atctcgactt tgaataaatt cacaagttct tctccatggc
atgcaccagt 300 cagattatga tcaccaaatg ttccttttgc aggatgactt
tccgagaagg aatattcata 360 ataataaact ggttcgtctt gtttctccac
agacaacttg acatgcttgt caacacctct 420 agtaaaccaa atatctccta
tcaagtcaat aaatttctgt tcattttctg gaacgggttt 480 atcgtcaaag
taaaacttcc tgattttttc tgcaagtttt ttcgattcct ctgatccatg 540
gactaattca aattcggctg gtacgagtct ttcaaaatct gcttcagctt ggttcagcat
600 ctctggtttt tctttcatga agaatttgaa caaaagtcct tctgcactgt
taaatccaac 660 taataaaggt actttgttaa aggatccgga tttcattctg
gccaatggtg attcttccaa 720 gaaaggttgg tgggatggga aaactttttc
aattgatggt acgaagacaa actcttctat 780 tatttcacct tctgtttcag
ctggcatttt cttcaggaga ctgtctacgg gggctttttg 840 taagaattct
aaagcatctt gagtattgtt tgtgggatga cccaatatct cagcaagttg 900
aagactacgc tttactggat gtctttggaa ggcccatgga ttaaaagcac ttccactttg
960 agcaattgcc cttttgtaaa gtccagtggt tgtatgagat aacaaaagat
aatgaacact 1020 tgctccacca gcagaaacac caccaattgt aatattttct
ggatctccac caaatttctc 1080 aatgttttct ttggtccatt tcagagctgc
cacctgatcc aataatccta catttcctgg 1140 agcaccctcc aactccaaat
tcagaaatcc gagaggtccc aatcggtaat tgaaagttac 1200 gaaaataaca
tcaaaatyta ctaaataatc tgggctttgg aattctaaat ttccggatcc 1260
agtcacaaaa ccaccaccat gaacccagaa aaatactgga agttttttat cagaagttgt
1320 ttttggtgcg tacacgttta ctaccaagca gtcttcgtct ccttcaattt
tcttcaagaa 1380 gaaatttaaa gatttacaca catttccgta taatgtggcg
ttgaaaacac cttgccatgg 1440 ctcagctttc tgtggaggct taaatctaag
ttctccaaca ggaggtttag cataaggtac 1500 acctgtgtag ctaaaatact
cttttccatt ttcgttcgtc aaagcttttc caaccaattc 1560 gccttggggc
aaagttacag tcggggaatc acattttata aaattaaaac taaacaaaaa 1620
aatacaactt aaaaaaataa cacgagacat 1650 62 29 DNA Artificial sequence
Synthetic Primer 62 aaactcgagt cccccgactg taactttgc 29 63 36 DNA
Artificial sequence Synthetic Primer 63 tcatctgcag ttattgactg
tgcaaagttt ttgtgg 36 64 32 DNA Artificial sequence Synthetic Primer
64 ttccggatcc ggctgatcta caagtgactt tg 32 65 34 DNA Artificial
sequence Synthetic Primer 65 tggtactcga gtcataaaaa tttattccaa aatc
34 66 39 DNA Artificial sequence Synthetic Primer 66 aaaactgcag
tataaatatg ttacctcaca gtgcattag 39 67 1987 DNA Ctenocephalides
felis CDS (231)..(1820) 67 aattcacagt gtaaataatt ttatttgata
taaatgtatt taatttttat tttaatctaa 60 ttttaattta aatatatata
gttttattta taaaaaaata ttttttttat gatcgaaaag 120 aaatttttat
ttatgtttat gagtgtgtgt tttggctatg atttacatta tttttgagct 180
agtataaaat taaaccatat tatattttgg atatataata acattttata atg tgt 236
Met Cys 1 gat cca tta cta aaa aca aca aca tat gga att ctg aaa ggc
aag aaa 284 Asp Pro Leu Leu Lys Thr Thr Thr Tyr Gly Ile Leu Lys Gly
Lys Lys 5 10 15 gtt gta aac gaa aat ggt aaa att tac tat agt tac aca
ggt ata ccc 332 Val Val Asn Glu Asn Gly Lys Ile Tyr Tyr Ser Tyr Thr
Gly Ile Pro 20 25 30 tat gca aaa tct cct gta aat gat ctc aga ttc
aag cca cca caa aaa 380 Tyr Ala Lys Ser Pro Val Asn Asp Leu Arg Phe
Lys Pro Pro Gln Lys 35 40 45 50 ctt gat cct tgg aat ggt gtt ttt gac
gcc act cag tat gga aat aat 428 Leu Asp Pro Trp Asn Gly Val Phe Asp
Ala Thr Gln Tyr Gly Asn Asn 55 60 65 tgt gct gct ggg aaa tgg ttt
ttg aaa tca gct ggg ggt tgc gaa gat 476 Cys Ala Ala Gly Lys Trp Phe
Leu Lys Ser Ala Gly Gly Cys Glu Asp 70 75 80 tgc ctt tac tta aat
atc tat gtc cca caa aac act tca gaa aat cct 524 Cys Leu Tyr Leu Asn
Ile Tyr Val Pro Gln Asn Thr Ser Glu Asn Pro 85 90 95 ttg cca gta
atg ttt tgg att cat gga gga gca ttt gtg gtc gga tca 572 Leu Pro Val
Met Phe Trp Ile His Gly Gly Ala Phe Val Val Gly Ser 100 105 110 gga
aat tct gat ata cat ggt cct gat tat tta ata gaa tat gat att 620 Gly
Asn Ser Asp Ile His Gly Pro Asp Tyr Leu Ile Glu Tyr Asp Ile 115 120
125 130 atc tta gta act att aat tat cgt cta gga cca ctt ggt ttt ctt
aat 668 Ile Leu Val Thr Ile Asn Tyr Arg Leu Gly Pro Leu Gly Phe Leu
Asn 135 140 145 ttg gaa atc gaa gat gcg cct ggg aat gtt gga ttg atg
gat caa gtt 716 Leu Glu Ile Glu Asp Ala Pro Gly Asn Val Gly Leu Met
Asp Gln Val 150 155 160 gca gcc cta aaa tgg gta aat gaa aat att gca
acc ttt agt gga gac 764 Ala Ala Leu Lys Trp Val Asn Glu Asn Ile Ala
Thr Phe Ser Gly Asp 165 170 175 cca aaa aat att aca att tgt gga gca
act gct gga gct gca agt gta 812 Pro Lys Asn Ile Thr Ile Cys Gly Ala
Thr Ala Gly Ala Ala Ser Val 180 185 190 cat tat cac att ttg tca caa
ctt acc aaa ggt tta ttc cac aag gct 860 His Tyr His Ile Leu Ser Gln
Leu Thr Lys Gly Leu Phe His Lys Ala 195 200 205 210 ata gca caa agt
gga agt gct ttt aat ccc tgg gct ttc caa aaa aat 908 Ile Ala Gln Ser
Gly Ser Ala Phe Asn Pro Trp Ala Phe Gln Lys Asn 215 220 225 cct gtt
aag aat gca ctt cga cta tgc aaa acc tta ggc ctt acc aca 956 Pro Val
Lys Asn Ala Leu Arg Leu Cys Lys Thr Leu Gly Leu Thr Thr 230 235 240
aac aac ctt caa gaa gcc ttg gat ttt ttg aaa aac cta cca gta gaa
1004 Asn Asn Leu Gln Glu Ala Leu Asp Phe Leu Lys Asn Leu Pro Val
Glu 245 250 255 aca ttg tta aat acc aaa tta ccc caa gaa att gat ggt
caa ctg ctg 1052 Thr Leu Leu Asn Thr Lys Leu Pro Gln Glu Ile Asp
Gly Gln Leu Leu 260 265 270 gat gac ttc gtg ttt gta cct tcg att gaa
aaa aca ttt cca gaa caa 1100 Asp Asp Phe Val Phe Val Pro Ser Ile
Glu Lys Thr Phe Pro Glu Gln 275 280 285 290 gat tcg tac tta act gac
ttg cca ata cca ata ata aat tca gga aaa 1148 Asp Ser Tyr Leu Thr
Asp Leu Pro Ile Pro Ile Ile Asn Ser Gly Lys 295 300 305 ttc cac aaa
gtt cca ttg ttg aca ggt tac aac agt gcc gaa ggc aat 1196 Phe His
Lys Val Pro Leu Leu Thr Gly Tyr Asn Ser Ala Glu Gly Asn 310 315 320
cta ttt ttc atg tac tta aaa aca gat cca gat tta tta aat aaa ttt
1244 Leu Phe Phe Met Tyr Leu Lys Thr Asp Pro Asp Leu Leu Asn Lys
Phe 325 330 335 gaa gct gat ttt gaa aga ttt ata cca act gac tta gaa
tta cct ttg 1292 Glu Ala Asp Phe Glu Arg Phe Ile Pro Thr Asp Leu
Glu Leu Pro Leu 340 345 350 cga tca caa aaa tct att gca ctg ggt gaa
gca atc agg gaa ttt tat 1340 Arg Ser Gln Lys Ser Ile Ala Leu Gly
Glu Ala Ile Arg Glu Phe Tyr 355 360 365 370 ttc caa aac aaa acc ata
tca gaa aat atg cag aat ttt gta gat gtt 1388 Phe Gln Asn Lys Thr
Ile Ser Glu Asn Met Gln Asn Phe Val Asp Val 375 380 385 tta agt gat
aat tgg ttt aca cgt gga att gat gag caa gta aag tta 1436 Leu Ser
Asp Asn Trp Phe Thr Arg Gly Ile Asp Glu Gln Val Lys Leu 390 395 400
act gtt aaa aat cag gaa gaa cca gtt ttt tat tat gtt tat aat ttt
1484 Thr Val Lys Asn Gln Glu Glu Pro Val Phe Tyr Tyr Val Tyr Asn
Phe 405 410 415 gat gaa aat tct cca agt cgg aaa gtt ttt ggt gat ttt
gga ata aaa 1532 Asp Glu Asn Ser Pro Ser Arg Lys Val Phe Gly Asp
Phe Gly Ile Lys 420 425 430 ggc ggt ggt cat gct gat gaa ttg ggt aat
ata ttt aaa gcc aaa agt 1580 Gly Gly Gly His Ala Asp Glu Leu Gly
Asn Ile Phe Lys Ala Lys Ser 435 440 445 450 gca aat ttt ggg aag gaa
aca cca aat gct gtg ttg gtt cag aga agg 1628 Ala Asn Phe Gly Lys
Glu Thr Pro Asn Ala Val Leu Val Gln Arg Arg 455 460 465 atg ctg gag
atg tgg act aat ttt gct aaa ttt gga aat cct act cca 1676 Met Leu
Glu Met Trp Thr Asn Phe Ala Lys Phe Gly Asn Pro Thr Pro 470 475 480
gct att acg gat aca ctt cca ata aaa tgg gaa cct gct ttt aaa gaa
1724 Ala Ile Thr Asp Thr Leu Pro Ile Lys Trp Glu Pro Ala Phe Lys
Glu 485 490 495 aat atg act ttt gtt caa att gac att gat tta aat ttg
agt act gat 1772 Asn Met Thr Phe Val Gln Ile Asp Ile Asp Leu Asn
Leu Ser Thr Asp 500 505 510 cca cta aaa agt cgt atg gaa ttt ggg aat
aaa ata aaa tta tta aaa 1820 Pro Leu Lys Ser Arg Met Glu Phe Gly
Asn Lys Ile Lys Leu Leu Lys 515 520 525 530 taagtaacta tacttagcta
aaccataata taccaaataa tagtatagga atacttcaca 1880 attttttgtt
acttcgttaa gtaaatttaa ttttttataa aaccaacttt tacgaataaa 1940
aaatgtaatt attttggaaa aaaaaaagaa aaaaaaaaaa aaaaaac 1987 68 530 PRT
Ctenocephalides felis 68 Met Cys Asp Pro Leu Leu Lys Thr Thr Thr
Tyr Gly Ile Leu Lys Gly 1 5 10 15 Lys Lys Val Val Asn Glu Asn Gly
Lys Ile Tyr Tyr Ser Tyr Thr Gly 20 25 30 Ile Pro Tyr Ala Lys Ser
Pro Val Asn Asp Leu Arg Phe Lys Pro Pro 35 40 45 Gln Lys Leu Asp
Pro Trp Asn Gly Val Phe Asp Ala Thr Gln Tyr Gly 50 55 60 Asn Asn
Cys Ala Ala Gly Lys Trp Phe Leu Lys Ser Ala Gly Gly Cys 65 70 75 80
Glu Asp Cys Leu Tyr Leu Asn Ile Tyr Val Pro Gln Asn Thr Ser Glu 85
90 95 Asn Pro Leu Pro Val Met Phe Trp Ile His Gly Gly Ala Phe Val
Val 100 105 110 Gly Ser Gly Asn Ser Asp Ile His Gly Pro Asp Tyr Leu
Ile Glu Tyr 115 120 125 Asp Ile Ile Leu Val Thr Ile Asn Tyr Arg Leu
Gly Pro Leu Gly Phe 130 135 140 Leu Asn Leu Glu Ile Glu Asp Ala Pro
Gly Asn Val Gly Leu Met Asp 145 150 155 160 Gln Val Ala Ala Leu Lys
Trp Val Asn Glu Asn Ile Ala Thr Phe Ser 165 170 175 Gly Asp Pro Lys
Asn Ile Thr Ile Cys Gly Ala Thr Ala Gly Ala Ala 180 185 190 Ser Val
His Tyr His Ile Leu Ser Gln Leu Thr Lys Gly Leu Phe His 195 200 205
Lys Ala Ile Ala Gln Ser Gly Ser Ala Phe Asn Pro Trp Ala Phe Gln 210
215 220 Lys Asn Pro Val Lys Asn Ala Leu Arg Leu Cys Lys Thr Leu Gly
Leu 225 230 235 240 Thr Thr Asn Asn Leu Gln Glu Ala Leu Asp Phe Leu
Lys Asn Leu Pro 245 250 255 Val Glu Thr Leu Leu Asn Thr Lys Leu Pro
Gln Glu Ile Asp Gly Gln 260 265 270 Leu Leu Asp Asp Phe Val Phe Val
Pro Ser Ile Glu Lys Thr Phe Pro 275 280 285 Glu Gln Asp Ser Tyr Leu
Thr Asp Leu Pro Ile Pro Ile Ile Asn Ser 290 295 300 Gly Lys Phe His
Lys Val Pro Leu Leu Thr Gly Tyr Asn Ser Ala Glu 305 310 315 320 Gly
Asn Leu Phe Phe Met Tyr Leu Lys Thr Asp Pro Asp Leu Leu Asn 325 330
335 Lys Phe Glu Ala Asp Phe Glu Arg Phe Ile Pro Thr Asp Leu Glu Leu
340 345 350 Pro Leu Arg Ser Gln Lys Ser Ile Ala Leu Gly Glu Ala Ile
Arg Glu 355 360 365 Phe Tyr Phe Gln Asn Lys Thr Ile Ser Glu Asn Met
Gln Asn Phe Val 370 375 380 Asp Val Leu Ser Asp Asn Trp Phe Thr Arg
Gly Ile Asp Glu Gln Val 385 390 395 400 Lys Leu Thr Val Lys Asn Gln
Glu Glu Pro Val Phe Tyr Tyr Val Tyr 405 410 415 Asn Phe Asp Glu Asn
Ser Pro Ser Arg Lys Val Phe Gly Asp Phe Gly 420 425 430 Ile Lys Gly
Gly Gly His Ala Asp Glu Leu Gly Asn Ile Phe Lys Ala 435 440 445 Lys
Ser Ala Asn Phe Gly Lys Glu Thr Pro Asn Ala Val Leu Val Gln 450 455
460 Arg Arg Met Leu Glu Met Trp Thr Asn Phe Ala Lys Phe Gly Asn Pro
465 470 475 480 Thr Pro Ala Ile Thr Asp Thr Leu Pro Ile Lys Trp Glu
Pro Ala Phe 485 490 495 Lys Glu Asn Met Thr Phe Val Gln Ile Asp Ile
Asp Leu Asn Leu Ser 500 505 510 Thr Asp Pro Leu Lys Ser Arg Met Glu
Phe Gly Asn Lys Ile Lys Leu 515 520 525 Leu Lys 530 69 1987 DNA
Ctenocephalides felis 69 gttttttttt tttttttttc tttttttttt
ccaaaataat tacatttttt attcgtaaaa 60 gttggtttta taaaaaatta
aatttactta acgaagtaac aaaaaattgt gaagtattcc 120 tatactatta
tttggtatat tatggtttag ctaagtatag ttacttattt taataatttt 180
attttattcc caaattccat acgacttttt agtggatcag tactcaaatt taaatcaatg
240 tcaatttgaa caaaagtcat attttcttta aaagcaggtt cccattttat
tggaagtgta 300 tccgtaatag ctggagtagg atttccaaat ttagcaaaat
tagtccacat ctccagcatc 360 cttctctgaa ccaacacagc atttggtgtt
tccttcccaa aatttgcact tttggcttta 420 aatatattac ccaattcatc
agcatgacca ccgcctttta ttccaaaatc accaaaaact 480 ttccgacttg
gagaattttc atcaaaatta taaacataat aaaaaactgg ttcttcctga 540
tttttaacag ttaactttac ttgctcatca attccacgtg taaaccaatt atcacttaaa
600 acatctacaa aattctgcat attttctgat atggttttgt tttggaaata
aaattccctg 660 attgcttcac ccagtgcaat agatttttgt gatcgcaaag
gtaattctaa gtcagttggt 720 ataaatcttt caaaatcagc ttcaaattta
tttaataaat ctggatctgt ttttaagtac 780 atgaaaaata gattgccttc
ggcactgttg taacctgtca acaatggaac tttgtggaat 840 tttcctgaat
ttattattgg tattggcaag tcagttaagt acgaatcttg ttctggaaat 900
gttttttcaa tcgaaggtac aaacacgaag tcatccagca gttgaccatc aatttcttgg
960 ggtaatttgg tatttaacaa tgtttctact ggtaggtttt tcaaaaaatc
caaggcttct 1020 tgaaggttgt ttgtggtaag gcctaaggtt ttgcatagtc
gaagtgcatt cttaacagga 1080 tttttttgga aagcccaggg attaaaagca
cttccacttt gtgctatagc cttgtggaat 1140 aaacctttgg taagttgtga
caaaatgtga taatgtacac ttgcagctcc agcagttgct 1200 ccacaaattg
taatattttt tgggtctcca ctaaaggttg caatattttc atttacccat 1260
tttagggctg caacttgatc catcaatcca acattcccag gcgcatcttc gatttccaaa
1320 ttaagaaaac caagtggtcc tagacgataa ttaatagtta ctaagataat
atcatattct 1380 attaaataat caggaccatg tatatcagaa tttcctgatc
cgaccacaaa tgctcctcca 1440 tgaatccaaa acattactgg caaaggattt
tctgaagtgt tttgtgggac atagatattt 1500 aagtaaaggc aatcttcgca
acccccagct gatttcaaaa accatttccc agcagcacaa 1560 ttatttccat
actgagtggc gtcaaaaaca ccattccaag gatcaagttt ttgtggtggc 1620
ttgaatctga gatcatttac aggagatttt gcatagggta tacctgtgta actatagtaa
1680 attttaccat tttcgtttac aactttcttg cctttcagaa ttccatatgt
tgttgttttt 1740 agtaatggat cacacattat aaaatgttat tatatatcca
aaatataata tggtttaatt 1800 ttatactagc tcaaaaataa tgtaaatcat
agccaaaaca cacactcata aacataaata 1860 aaaatttctt ttcgatcata
aaaaaaatat ttttttataa ataaaactat atatatttaa 1920 attaaaatta
gattaaaata aaaattaaat acatttatat caaataaaat tatttacact 1980 gtgaatt
1987 70 1590 DNA Ctenocephalides felis exon (1)..(1590) 70 atg tgt
gat cca tta cta aaa aca aca aca tat gga att ctg aaa ggc 48 Met Cys
Asp Pro Leu Leu Lys Thr Thr Thr Tyr Gly Ile Leu Lys Gly 1 5 10 15
aag aaa gtt gta aac gaa aat ggt aaa att tac tat agt tac aca ggt 96
Lys Lys Val Val Asn Glu Asn Gly Lys Ile Tyr Tyr Ser Tyr Thr Gly 20
25 30 ata ccc tat gca aaa tct cct gta aat gat ctc aga ttc aag cca
cca 144 Ile Pro Tyr Ala Lys Ser Pro Val Asn Asp Leu Arg Phe Lys Pro
Pro 35 40 45 caa aaa ctt gat cct tgg aat ggt gtt ttt gac gcc act
cag tat gga 192 Gln Lys Leu Asp Pro Trp Asn Gly Val Phe Asp Ala Thr
Gln Tyr Gly 50 55 60 aat aat tgt gct gct ggg aaa tgg ttt ttg aaa
tca gct ggg ggt tgc 240 Asn Asn Cys Ala Ala Gly Lys Trp Phe Leu Lys
Ser Ala Gly Gly Cys 65 70 75 80 gaa gat tgc ctt tac tta aat atc tat
gtc cca caa aac act tca gaa 288 Glu Asp Cys Leu Tyr Leu Asn Ile Tyr
Val Pro Gln Asn Thr Ser Glu 85 90 95 aat cct ttg cca gta atg ttt
tgg att cat gga gga gca ttt gtg gtc 336 Asn Pro Leu Pro Val Met Phe
Trp Ile His Gly Gly Ala Phe Val Val 100 105 110 gga tca gga aat tct
gat ata cat ggt cct gat tat tta ata gaa tat 384 Gly Ser Gly Asn Ser
Asp Ile His Gly Pro Asp Tyr Leu Ile Glu Tyr 115 120 125 gat att atc
tta gta act att aat tat cgt cta gga cca ctt ggt ttt 432 Asp Ile Ile
Leu Val Thr Ile Asn Tyr Arg Leu Gly Pro Leu Gly Phe 130 135 140 ctt
aat ttg gaa atc gaa gat gcg cct ggg aat gtt gga ttg atg gat 480 Leu
Asn Leu Glu Ile Glu Asp Ala Pro Gly Asn Val Gly Leu Met Asp 145 150
155 160 caa gtt gca gcc cta aaa tgg gta aat gaa aat att gca acc ttt
agt 528 Gln Val Ala Ala Leu Lys Trp Val Asn Glu Asn Ile Ala Thr Phe
Ser 165 170 175 gga gac cca aaa aat att aca att tgt gga gca act gct
gga gct gca 576 Gly Asp Pro Lys Asn Ile Thr Ile Cys Gly Ala Thr Ala
Gly Ala Ala 180 185 190 agt gta cat tat cac att ttg tca caa ctt acc
aaa ggt tta ttc cac 624 Ser Val His Tyr His Ile Leu Ser Gln Leu Thr
Lys Gly Leu Phe His 195 200 205 aag gct ata gca caa agt gga agt gct
ttt aat ccc tgg gct ttc caa 672 Lys Ala Ile Ala Gln Ser Gly Ser Ala
Phe Asn Pro Trp Ala Phe Gln 210 215 220 aaa aat cct gtt aag aat gca
ctt cga cta tgc aaa acc tta ggc ctt 720 Lys Asn Pro Val Lys Asn Ala
Leu Arg Leu Cys Lys Thr Leu Gly Leu 225 230 235 240 acc aca aac aac
ctt caa gaa gcc ttg gat ttt ttg aaa aac cta cca 768 Thr Thr Asn Asn
Leu Gln Glu Ala Leu Asp Phe Leu Lys Asn Leu Pro 245 250 255 gta gaa
aca ttg tta aat acc aaa tta ccc caa gaa att gat ggt caa 816 Val Glu
Thr Leu Leu Asn Thr Lys Leu Pro Gln Glu Ile Asp Gly Gln 260 265 270
ctg ctg gat gac ttc gtg ttt gta cct tcg att gaa aaa aca ttt cca 864
Leu Leu Asp Asp Phe Val Phe Val Pro Ser Ile Glu Lys Thr Phe Pro 275
280 285 gaa caa gat tcg tac tta act gac ttg cca ata cca ata ata aat
tca 912 Glu Gln Asp Ser Tyr Leu Thr Asp Leu Pro Ile Pro Ile Ile Asn
Ser 290 295 300 gga aaa ttc cac aaa gtt cca ttg ttg aca ggt tac aac
agt gcc gaa 960 Gly Lys Phe His Lys Val Pro Leu Leu Thr Gly Tyr Asn
Ser Ala Glu 305 310 315 320 ggc aat cta ttt ttc atg tac tta aaa aca
gat cca gat tta tta aat 1008 Gly Asn Leu Phe Phe Met Tyr Leu Lys
Thr Asp Pro Asp Leu Leu Asn 325 330 335 aaa ttt gaa gct gat ttt gaa
aga ttt ata cca act gac tta gaa tta 1056 Lys Phe Glu Ala Asp Phe
Glu Arg Phe Ile Pro Thr Asp Leu Glu Leu 340 345 350 cct ttg cga tca
caa aaa tct att gca ctg ggt gaa gca atc agg gaa 1104 Pro Leu Arg
Ser Gln Lys Ser Ile Ala Leu Gly Glu Ala Ile Arg Glu 355 360 365 ttt
tat ttc caa aac aaa acc ata tca gaa aat atg cag aat ttt gta 1152
Phe Tyr Phe Gln Asn Lys Thr Ile Ser Glu Asn Met Gln Asn Phe Val 370
375 380 gat gtt tta agt gat aat tgg ttt aca cgt gga att gat gag caa
gta 1200 Asp Val Leu Ser Asp Asn Trp Phe Thr Arg Gly Ile Asp Glu
Gln Val 385 390 395 400 aag tta act gtt aaa aat cag gaa gaa cca gtt
ttt tat tat gtt tat 1248 Lys Leu Thr Val Lys Asn Gln Glu Glu Pro
Val Phe Tyr Tyr Val Tyr 405 410 415 aat ttt gat gaa aat tct cca agt
cgg aaa gtt ttt ggt gat ttt gga 1296 Asn Phe Asp Glu Asn Ser Pro
Ser Arg Lys Val Phe Gly Asp Phe Gly 420 425 430 ata aaa ggc ggt ggt
cat gct gat gaa ttg ggt aat ata ttt aaa gcc 1344 Ile Lys Gly Gly
Gly His Ala Asp Glu Leu Gly Asn Ile Phe Lys Ala 435 440 445 aaa agt
gca aat ttt ggg aag gaa aca cca aat gct gtg ttg gtt cag 1392 Lys
Ser Ala Asn Phe Gly Lys Glu Thr Pro Asn Ala Val Leu Val Gln 450 455
460 aga agg atg ctg gag atg tgg act aat ttt gct aaa ttt gga aat cct
1440 Arg Arg Met Leu Glu Met Trp Thr Asn Phe Ala Lys Phe Gly Asn
Pro 465 470 475 480 act cca gct att acg gat aca ctt cca ata aaa tgg
gaa cct gct ttt 1488 Thr Pro Ala Ile Thr Asp Thr Leu Pro Ile Lys
Trp Glu Pro Ala Phe 485 490 495 aaa gaa aat atg act ttt gtt caa att
gac att gat tta aat ttg agt 1536 Lys Glu Asn Met Thr Phe Val Gln
Ile Asp Ile Asp Leu Asn Leu Ser 500 505 510 act gat cca cta aaa agt
cgt atg gaa ttt ggg aat aaa ata aaa tta 1584 Thr Asp Pro Leu Lys
Ser Arg Met Glu Phe Gly Asn Lys Ile Lys Leu 515 520 525 tta aaa
1590 Leu Lys 530 71 1590 DNA Ctenocephalides felis 71 ttttaataat
tttattttat tcccaaattc catacgactt tttagtggat cagtactcaa 60
atttaaatca atgtcaattt gaacaaaagt catattttct ttaaaagcag gttcccattt
120 tattggaagt gtatccgtaa tagctggagt aggatttcca aatttagcaa
aattagtcca 180 catctccagc atccttctct gaaccaacac agcatttggt
gtttccttcc caaaatttgc 240 acttttggct ttaaatatat tacccaattc
atcagcatga ccaccgcctt ttattccaaa 300 atcaccaaaa actttccgac
ttggagaatt ttcatcaaaa ttataaacat aataaaaaac 360 tggttcttcc
tgatttttaa cagttaactt tacttgctca tcaattccac gtgtaaacca 420
attatcactt aaaacatcta caaaattctg catattttct gatatggttt tgttttggaa
480 ataaaattcc ctgattgctt cacccagtgc aatagatttt tgtgatcgca
aaggtaattc 540 taagtcagtt ggtataaatc tttcaaaatc agcttcaaat
ttatttaata aatctggatc 600 tgtttttaag tacatgaaaa atagattgcc
ttcggcactg ttgtaacctg tcaacaatgg 660 aactttgtgg aattttcctg
aatttattat tggtattggc aagtcagtta agtacgaatc 720 ttgttctgga
aatgtttttt caatcgaagg tacaaacacg aagtcatcca gcagttgacc 780
atcaatttct tggggtaatt tggtatttaa caatgtttct actggtaggt ttttcaaaaa
840 atccaaggct tcttgaaggt tgtttgtggt aaggcctaag gttttgcata
gtcgaagtgc 900 attcttaaca ggattttttt ggaaagccca gggattaaaa
gcacttccac tttgtgctat 960 agccttgtgg aataaacctt tggtaagttg
tgacaaaatg tgataatgta cacttgcagc 1020 tccagcagtt gctccacaaa
ttgtaatatt ttttgggtct ccactaaagg ttgcaatatt 1080 ttcatttacc
cattttaggg ctgcaacttg atccatcaat ccaacattcc caggcgcatc 1140
ttcgatttcc aaattaagaa aaccaagtgg tcctagacga taattaatag ttactaagat
1200 aatatcatat tctattaaat aatcaggacc atgtatatca gaatttcctg
atccgaccac 1260 aaatgctcct ccatgaatcc aaaacattac tggcaaagga
ttttctgaag tgttttgtgg 1320 gacatagata tttaagtaaa ggcaatcttc
gcaaccccca gctgatttca aaaaccattt 1380 cccagcagca caattatttc
catactgagt ggcgtcaaaa acaccattcc aaggatcaag 1440 tttttgtggt
ggcttgaatc tgagatcatt tacaggagat tttgcatagg gtatacctgt 1500
gtaactatag taaattttac cattttcgtt tacaactttc ttgcctttca gaattccata
1560 tgttgttgtt tttagtaatg gatcacacat 1590 72 650 DNA
Ctenocephalides felis CDS (3)..(650) 72 gg atc cat gga ggc gca ttc
aac caa gga tca gga tct tat aat ttt 47 Ile His Gly Gly Ala Phe Asn
Gln Gly Ser Gly Ser Tyr Asn Phe 1 5 10 15 ttt gga cct gat tat ttg
atc agg gaa gga att att ttg gtc act atc 95 Phe Gly Pro Asp Tyr Leu
Ile Arg Glu Gly Ile Ile Leu Val Thr Ile 20 25 30 aac tat aga tta
gga gtt ttc ggt ttt cta tca gcg ccg gaa tgg gat 143 Asn Tyr Arg Leu
Gly Val Phe Gly Phe Leu Ser Ala Pro Glu Trp Asp 35 40 45 atc cat
gga aat atg ggt cta aaa gac cag aga ttg gca cta aaa tgg 191 Ile His
Gly Asn Met Gly Leu Lys Asp Gln Arg Leu Ala Leu Lys Trp 50 55 60
gtt tac gac aac atc gaa aag ttt ggt gga gac aga gaa aaa att aca 239
Val Tyr Asp Asn Ile Glu Lys Phe Gly Gly Asp Arg Glu Lys Ile Thr 65
70 75 att gct gga gaa tct gct gga gca gca agt gtc cat ttt ctg atg
atg 287 Ile Ala Gly Glu Ser Ala Gly Ala Ala Ser Val His Phe Leu Met
Met 80 85 90 95 gac aac tcg act aga aaa tac tac caa agg gcc att ttg
cag agt ggg 335 Asp Asn Ser Thr Arg Lys Tyr Tyr Gln Arg Ala Ile Leu
Gln Ser Gly 100 105 110 aca tta cta aat ccg act gct aat caa att caa
ctt ctg cat aga ttt 383 Thr Leu Leu Asn Pro Thr Ala Asn Gln Ile Gln
Leu Leu His Arg Phe 115 120 125 gaa aaa ctc aaa caa gtg cta aac atc
acg caa aaa caa gaa ctc cta 431 Glu Lys Leu Lys Gln Val Leu Asn Ile
Thr Gln Lys Gln Glu Leu Leu 130 135 140 aac ctg gat aaa aac cta att
tta cga gca gcc tta aac aga gtt cct 479 Asn Leu Asp Lys Asn Leu Ile
Leu Arg Ala Ala Leu Asn Arg Val Pro 145 150 155 gat agc aac gac cat
gac cga gac aca gta cca gta ttt aat cca gtc 527 Asp Ser Asn Asp His
Asp Arg Asp Thr Val Pro Val Phe Asn Pro Val 160 165 170 175 tta gaa
tca cca gaa tct cca gat cca ata aca ttt cca tct gcc ttg 575 Leu Glu
Ser Pro Glu Ser Pro Asp Pro Ile Thr Phe Pro Ser Ala Leu 180 185 190
gaa aga atg aga aat ggt gaa ttt cct gat gtc gat gtc atc att ggt 623
Glu Arg Met Arg Asn Gly Glu Phe Pro Asp Val Asp Val Ile Ile Gly 195
200 205 ttc aat agt gct gaa ggt tta aga tct 650 Phe Asn Ser Ala Glu
Gly Leu Arg Ser 210 215 73 216 PRT Ctenocephalides felis 73 Ile His
Gly Gly Ala Phe Asn Gln Gly Ser Gly Ser Tyr Asn Phe Phe 1 5 10 15
Gly Pro Asp Tyr Leu Ile Arg Glu Gly Ile Ile Leu Val Thr Ile Asn 20
25 30 Tyr Arg Leu Gly Val Phe Gly Phe Leu Ser Ala Pro Glu Trp Asp
Ile 35 40 45 His Gly Asn Met Gly Leu Lys Asp Gln Arg Leu Ala Leu
Lys Trp Val 50 55 60 Tyr Asp Asn Ile Glu Lys Phe Gly Gly Asp Arg
Glu Lys Ile Thr Ile 65 70 75 80 Ala Gly Glu Ser Ala Gly Ala Ala Ser
Val His Phe Leu Met Met Asp 85 90 95 Asn Ser Thr Arg Lys Tyr Tyr
Gln Arg Ala Ile Leu Gln Ser Gly Thr 100 105 110 Leu Leu Asn Pro Thr
Ala Asn Gln Ile Gln Leu Leu His Arg Phe Glu 115 120 125 Lys Leu Lys
Gln Val Leu Asn Ile Thr Gln Lys Gln Glu Leu Leu Asn 130 135 140 Leu
Asp Lys Asn Leu Ile Leu Arg Ala Ala Leu Asn Arg Val Pro Asp 145 150
155 160 Ser Asn Asp His Asp Arg Asp Thr Val Pro Val Phe Asn Pro Val
Leu 165 170 175 Glu Ser Pro Glu Ser Pro Asp Pro Ile Thr Phe Pro Ser
Ala Leu Glu 180 185 190 Arg Met Arg Asn Gly Glu Phe Pro Asp Val Asp
Val Ile Ile Gly Phe 195 200 205 Asn Ser Ala Glu Gly Leu Arg Ser 210
215 74 15 PRT Peptide MISC_FEATURE (3)..(3) Xaa = unknown 74 Asp
Leu Xaa Val Xaa Xaa Leu Gln Gly Thr Leu Lys Gly Lys Glu 1 5 10 15
75 31 DNA Artificial sequence Synthetic Primer 75 cgcggatccg
ctgatctaca agtgactttg c 31 76 1488 DNA Ctenocephalides felis exon
(3)..(1487) 76 cc cag ggc gaa ttg gtt gga aaa gct ttg acg aac gaa
aat gga aaa 47 Gln Gly Glu Leu Val Gly Lys Ala Leu Thr Asn Glu Asn
Gly Lys 1 5 10 15 gag tat ttt agc tac aca ggt gtg cct tat gct aaa
cct cca gtt gga 95 Glu Tyr Phe Ser Tyr Thr Gly Val Pro Tyr Ala Lys
Pro Pro Val Gly 20 25 30 gaa ctt aga ttt aag cct cca cag aaa gct
gag cca tgg aat ggt gtt 143 Glu Leu Arg Phe Lys Pro Pro Gln Lys Ala
Glu Pro Trp Asn Gly Val 35 40 45 ttc aac gcc aca tca cat gga aat
gtg tgc aaa gct ttg aat ttc ttc 191 Phe Asn Ala Thr Ser His Gly Asn
Val Cys Lys Ala Leu Asn Phe Phe 50 55 60 ttg aaa aaa att gaa gga
gac gaa gac tgc ttg ttg gtg aat gtg tac 239 Leu Lys Lys Ile Glu Gly
Asp Glu Asp Cys Leu Leu Val Asn Val Tyr 65 70 75 gca cca aaa aca
act tct gac aaa aaa ctt cca gta ttt ttc tgg gtt 287 Ala Pro Lys Thr
Thr Ser Asp Lys Lys Leu Pro Val Phe Phe Trp Val 80 85 90 95 cat ggt
ggc ggt ttt gtg act gga tcc gga aat tta gaa ttt caa agc 335 His Gly
Gly Gly Phe Val Thr Gly Ser Gly Asn Leu Glu Phe Gln Ser 100 105 110
cca gat tat tta gta aat tat gat gtt att ttt gta act ttc aat tac 383
Pro Asp Tyr Leu Val Asn Tyr Asp Val Ile Phe Val Thr Phe Asn Tyr 115
120 125 cga ttg gga cca ctc gga ttt ttg aat ttg gag ttg gaa ggt gct
cct 431 Arg Leu Gly Pro Leu Gly Phe Leu Asn Leu Glu Leu Glu Gly Ala
Pro 130 135 140 gga aat gta gga tta ttg gat cag gta gca gct ttg aaa
tgg acc aaa 479 Gly Asn Val Gly Leu Leu Asp Gln Val Ala Ala Leu Lys
Trp Thr Lys 145 150 155 gaa aat att gag aaa ttt ggt gga gat cca gaa
aat att aca att ggt 527 Glu Asn Ile Glu Lys Phe Gly Gly Asp Pro Glu
Asn Ile Thr Ile Gly 160 165 170 175 ggt gtt tct gct ggt gga gca agt
gtt cat tat ctt tta ttg tca cat 575 Gly Val Ser Ala Gly Gly Ala Ser
Val His Tyr Leu Leu Leu Ser His 180 185 190 aca acc act gga ctt tac
aaa agg gca att gct caa agt gga agt gct 623 Thr Thr Thr Gly Leu Tyr
Lys Arg Ala Ile Ala Gln Ser Gly Ser Ala 195 200 205 tta aat cca tgg
gcc ttc caa aga cat cca gta aag cgt agt ctt caa 671 Leu Asn Pro Trp
Ala Phe Gln Arg His Pro Val Lys Arg Ser Leu Gln 210 215 220 ctt gct
gag ata tta ggt cat ccc aca aac aac act caa gat gct tta 719 Leu Ala
Glu Ile Leu Gly His Pro Thr Asn Asn Thr Gln Asp Ala Leu 225 230 235
gaa ttc tta caa aaa gcc cca gta gac agt ctc ctg aaa aaa atg cca 767
Glu Phe Leu Gln Lys Ala Pro Val Asp Ser Leu Leu Lys Lys Met Pro 240
245 250 255 gct gaa aca gaa ggt gaa ata ata gaa gag ttc gtc ttc gta
cca tca 815 Ala Glu Thr Glu Gly Glu Ile Ile Glu Glu Phe Val Phe Val
Pro Ser 260 265 270 att gaa aaa gtt ttc cca tcc cac caa cct ttc ttg
gaa gaa tca cca 863 Ile Glu Lys Val Phe Pro Ser His Gln Pro Phe Leu
Glu Glu Ser Pro 275 280 285 ttg gcc aga atg aaa tct gga tcc ttt aac
aaa gta cct tta tta gtt 911 Leu Ala Arg Met Lys Ser Gly Ser Phe Asn
Lys Val Pro Leu Leu Val 290 295 300 gga ttc aac agc gca gaa gga ctt
ttg tac aaa ttc ttt atg aaa gaa 959 Gly Phe Asn Ser Ala Glu Gly Leu
Leu Tyr Lys Phe Phe Met Lys Glu 305 310 315 aaa cca gag atg ctg aac
caa gct gaa gca gat ttc gaa aga ctc gta 1007 Lys Pro Glu Met Leu
Asn Gln Ala Glu
Ala Asp Phe Glu Arg Leu Val 320 325 330 335 cca gcc gaa ttt gaa tta
gcc cat gga tca gaa gaa tcg aaa aaa ctt 1055 Pro Ala Glu Phe Glu
Leu Ala His Gly Ser Glu Glu Ser Lys Lys Leu 340 345 350 gca gaa aaa
atc agg aag ttt tac ttt gac gat aaa ccc gtt cct gaa 1103 Ala Glu
Lys Ile Arg Lys Phe Tyr Phe Asp Asp Lys Pro Val Pro Glu 355 360 365
aat gag cag aaa ttt att gac ttg ata gga gat att tgg ttt act aga
1151 Asn Glu Gln Lys Phe Ile Asp Leu Ile Gly Asp Ile Trp Phe Thr
Arg 370 375 380 ggc att gac aag cat gtc aag ttg tct gta gaa aaa caa
gac gag cca 1199 Gly Ile Asp Lys His Val Lys Leu Ser Val Glu Lys
Gln Asp Glu Pro 385 390 395 gta tat tat tat gaa tat tct ttc tct gaa
agt cat cct gca aaa gga 1247 Val Tyr Tyr Tyr Glu Tyr Ser Phe Ser
Glu Ser His Pro Ala Lys Gly 400 405 410 415 aca ttt ggt gac cat aac
ttg act gga gca tgt cat ggt gaa gaa ctt 1295 Thr Phe Gly Asp His
Asn Leu Thr Gly Ala Cys His Gly Glu Glu Leu 420 425 430 gtg aat tta
ttc aaa gtc gag atg atg aag ctg gaa aaa gat aaa ccg 1343 Val Asn
Leu Phe Lys Val Glu Met Met Lys Leu Glu Lys Asp Lys Pro 435 440 445
aat gtt tta tta aca aaa gat agg gta ctt gct atg tgg acg aac ttc
1391 Asn Val Leu Leu Thr Lys Asp Arg Val Leu Ala Met Trp Thr Asn
Phe 450 455 460 atc aaa aat gga aat cct act cct gaa gta act gaa tta
ttg cca gtt 1439 Ile Lys Asn Gly Asn Pro Thr Pro Glu Val Thr Glu
Leu Leu Pro Val 465 470 475 aaa tgg gaa cct gcc aca aaa gac aag ttg
aat tat ttg aac att gat g 1488 Lys Trp Glu Pro Ala Thr Lys Asp Lys
Leu Asn Tyr Leu Asn Ile Asp 480 485 490 495
* * * * *