AU2007221739A1 - Flea peritrophin nucleic acid molecules, proteins and uses thereof - Google Patents

Description

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AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION Standard Patent Applicant: HESKA CORPORATION Invention Title: FLEA PERITROPHIN NUCLEIC ACID MOLECULES, PROTEINS AND USES

THEREOF

The following statement is a full description of this invention, including the best method for performing it known to us: la- O FLEA PERITROPHIN NUCLEIC ACID MOLECULES, PROTEINS AND USES THEREOF 2 FIELD OF THE INVENTION The present invention relates to flea peritrophin 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 methods to obtain such proteins, nucleic acid molecules, antibodies, and inhibitory compounds. The present invention also includes therapeutic compositions comprising such inhibitors, as well as uses thereof.

(1 The entire disclosure in the complete specification of our Australian Patent Application No. 2001296834 is by this cross-reference incorporated into the present specification.

BACKGROUND OF THE INVENTION All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art.

The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

Flea infestation of animals is a health and economic concern for pet owners. In particular, the bites of fleas are a problem for animals maintained as pets because the infestation becomes a source of annoyance not only for the pet but also for the pet owner who may find his or her home generally contaminated with insects. Fleas also directly cause a variety of diseases, including allergy, and also carry a variety of infectious agents including, but not limited to, endoparasites nematodes, cestodes, trematodes and protozoa), bacteria and viruses. As such, fleas are a problem not only when they are on an animal but also when they are in the general environment of the animal.

The medical importance of flea infestation has prompted the development of reagents capable of controlling flea infestation. Commonly encountered methods to control flea infestation are generally focused on use of insecticides, which are often unsuccessful for one or more of the following reasons: failure of owner compliance (frequent administration is required); behavioral or physiological intolerance of the pet to the pesticide product or means N Wtccoume\Cases\Palent\480OA999\P485O4.AU\Specis\P48504 Claimsdoc 3 Aphil 2007 lb- 0 0 of administration; and the emergence of flea populations resistant to the prescribed dose of pesticide.

r) Peritrophins, including flea PL1, PL2, PL3, PL4 and PL5 proteins of the present 00 invention, are a family of putative chitin-binding proteins that comprise a structural component of the peritrophic matrix, an acellular membrane composed of proteins and sugars, most commonly chitin which forms a barrier between the contents of an ingested meal and the gut epithelia. Peritrophin-like proteins have also been epithelia. Peritrophin-like proteins have also been N:Mielboume\Cases\Patentl48000-48999\P48504 AU\Specis\P48504 Claims do 13 April 2007 0 shown to be present in the trachea of Drosophila embryos, indicating that such proteins may have additional roles outside the midgut. The function of the Speritrophin-like proteins in adult fleas is not clear, since adult fleas do not produce a 00 peritrophic matrix in the gut. Peritrophins have been investigated as targets for immunological control of hematophagous insects including the sheep blowfly, Lucilia e cuprina. It has been shown in this insect that ingestion of antibodies against r peritrophins inhibits the growth of larvae and can result in increased larval mortality.

It has also been shown that the ingestion of antibodies against peritrophins reduces the Spermeability of the peritrophic matrix in L. cuprina larvae. This in turn may inhibit the movement of digested food across the peritrophic matrix to the gut epithelium, resulting in starvation. As such, a flea peritrophin of the present invention represents a novel target for anti-flea vaccines and chemotherapeutic drugs.

Therefore, isolation and sequencing of flea peritrophin genes may be critical for use in identifying specific agents for treating animals for flea infestation.

SUMMARY OF THE INVENTION The present invention provides flea peritrophin nucleic acid molecules, proteins encoded by such nucleic acid molecules; antibodies raised against such proteins anti-flea peritrophin antibodies); mimetopes of such proteins or antibodies; compositions comprising such nucleic acid molecules, proteins, antibodies, and mimetopes; and compounds that inhibit flea peritrophin activity inhibitory compounds or inhibitors).

The present invention also includes methods to obtain such proteins, mimetopes, nucleic acid molecules, antibodies and inhibitory compounds. The present invention also includes the use of proteins and antibodies to identify such inhibitory compounds as well as assay kits to identify such inhibitory compounds. Also included in the present invention are therapeutic compositions comprising proteins, mimetopes, nucleic acid molecules, antibodies and inhibitory compounds of the present invention including therapeutic compounds derived from a protein of the present invention that inhibit the activity of flea peritrophin proteins; also included are uses of such therapeutic compounds.

One embodiment of the present invention is an isolated flea peritrophin nucleic a acid molecule that hybridizes with a nucleic acid sequence having SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, 0 SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID SNO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID r_ 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:37, SEQ ID SNO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and/or SEQ ID NO:49, under conditions that allow less than or equal to 30% base pair mismatch. Another embodiment of the present invention is an isolated flea peritrophin nucleic acid molecule having a nucleic acid sequence that is at least 70% identical to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID SEQ ID NO: 16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, 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:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID SEQ ID NO:46, SEQ ID NO:47, and/or SEQ ID NO:49 and fragments of any of such nucleic acid sequences of at least 35 nucleotides in length.

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.

Another embodiment of the present invention includes an isolated flea peritrophin protein that is at least 70% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO: 12, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:40, SEQ ID NO:43, and/or SEQ ID NO:48 and fragments thereof having at least 10 amino acid residues, wherein such fragments can O elicit an immune response against respective flea peritrophin proteins or selectively binds to an antibody that binds any of such amino acid sequences.

Another embodiment of the present invention includes an isolated flea peritrophin protein 00 encoded by a nucleic acid molecule at least 35 nucleotides in length that hybridizes with a nucleic acid sequence having SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:46, and/or SEQ ID NO:49, under conditions that allow less than or equal to 30% base pair mismatch.

Another embodiment of the present invention includes a composition comprising an C 1 excipient and a compound selected from the group consisting of nucleic acid molecules, proteins, and antibodies of the present invention and a method to treat an animal for flea infestation comprising administering such a composition to such an animal.

Another embodiment of the present invention includes a method to detect an inhibitor of flea peritrophin activity, said method comprising contacting an isolated flea peritrophin protein of the present invention, with a putative inhibitory compound under conditions in which, in the absence of said compound, said protein has flea peritrophin protein activity, and (b) determining if said putative inhibitory compound inhibits flea peritrophin protein activity.

DETAILED DESCRIPTION OF THE INVENTION In the claims which follow and in the description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e.

to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

The present invention provides for flea peritrophin nucleic acid molecules, proteins encoded by such nucleic acid molecules, antibodies raised against such proteins, and inhibitors of such proteins. As used herein, flea peritrophin nucleic acid molecules and proteins encoded by such nucleic acid molecules are also referred to as peritrophin nucleic acid molecules and proteins, or PL nucleic acid molecules and PL proteins, respectively. Flea peritrophin nucleic acid molecules and proteins of the present invention can be isolated from a flea or prepared recombinantly or synthetically. Flea peritrophin nucleic acid molecules of the present invention N \Melboun eU\ws\Patmct\4800-499'P48SO4A1DSpecisT48504 Claimd 3 Apil 2007 -4acan be RNA or DNA, or modified forms thereof, and can be double-stranded or single-stranded; examples of nucleic acid molecules include, but are not limited to, 00 N \Melboume\Cass alIenW8Oo.48999\P48504 AUI\SpeciS\P48S04 Claims dm 13 April 2007 O complementary DNA (cDNA) molecules, genomic DNA molecules, synthetic DNA molecules, DNA molecules which are specific tags for messenger RNA, and corresponding mRNA molecules. As such, a flea nucleic acid molecule of the present 00 invention is not intended refer to an entire chromosome within which such a nucleic acid molecule is contained, however, a flea peritrophin cDNA of the present invention Smay include all regions such as regulatory regions that control production of flea r_ peritrophin proteins encoded by such a cDNA (such as, but not limited to, Stranscription, translation or post-translation control regions) as well as the coding region itself, and any introns or non-translated coding regions. As used herein, the 0 10 phrase "flea peritrophin protein" refers to a protein encoded by a flea peritrophin nucleic acid molecule.

Peritrophins, including flea PL1, PL2, PL3, PL4 and PL5 proteins of the present invention, are a family of putative chitin-binding proteins that comprise a structural component of the peritrophic matrix, an acellular membrane composed of proteins and sugars, most commonly chitin which forms a barrier between the contents of an ingested meal and the gut epithelia. Flea peritrophin proteins of the present invention are characterized as containing a 6 cysteine motif, including a highly conserved motif at cysteine 2 through cysteine 3 of "CNNYYNC", where "N" represents any amino acid residue and represents an aromatic amino acid residue.

Flea peritrophin nucleic acid molecules of known length isolated from a flea, such as Ctenocephalidesfelis are denoted "nCfPL1,", for example nCfPLl10 6 wherein refers to the number of nucleotides in that molecule, and flea peritrophin proteins of known length are denoted "PCfPL1," (for example PCfPL127) wherein refers to the number of amino acid residues in that molecule.

The present invention also provides for flea peritrophin DNA molecules that are specific tags for messenger RNA molecules. Such DNA molecules can correspond to an entire or partial sequence of a messenger RNA, and therefore, a DNA molecule corresponding to such a messenger RNA molecule a cDNA molecule), can encode a full-length or partial-length protein. A nucleic acid molecule encoding a partiallength protein can be used directly as a probe or indirectly to generate primers to identify and/or isolate a cDNA nucleic acid molecule encoding a corresponding, or 0 structurally related, full-length protein. Such a partial cDNA nucleic acid molecule can also be used in a similar manner to identify a genomic nucleic acid molecule, such Vas a nucleic acid molecule that contains the complete gene including regulatory 00 regions, exons and introns. Methods for using partial flea peritrophin cDNA molecules and sequences to isolate full-length and corresponding cDNA molecules are described in the examples herein below.

e¢3 The proteins and nucleic acid molecules of the present invention can be obtained from their natural source, or can be produced using, for example, recombinant nucleic acid technology or chemical synthesis. Also included in the present invention is the use of these proteins and nucleic acid molecules as well as antibodies and inhibitory compounds thereto as therapeutic compositions to protect animals from flea infestation as well as in other applications, such as those disclosed below.

One embodiment of the present invention is an isolated protein that includes a flea peritrophin protein. It is to be noted that the term or "an" entity refers to one or more of that entity; for example, a protein, a nucleic acid molecule, an antibody and a therapeutic composition refers to "one or more" or "at least one" protein, nucleic acid molecule, antibody and therapeutic composition respectively. As such, the terms (or "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably. According to the present invention, an isolated, or biologically pure, protein, is a protein that has been removed from its natural milieu. As such, "isolated" and "biologically pure" do not necessarily reflect the extent to which the protein has been purified. An isolated protein of the present invention can be obtained from its natural source, can be produced using recombinant DNA technology, or can be produced by chemical synthesis.

As used herein, isolated flea peritrophin proteins of the present invention can be full-length proteins or any homologue of such proteins. An isolated protein of the present invention, including a homologue, can be identified in a straight-forward manner by the protein's ability to elicit an immune response against a flea peritrophin protein or by the protein's ability to exhibit flea peritrophin activity. Examples of flea O peritrophin homologue proteins include flea peritrophin proteins in which amino acids have been deleted a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized by glycosylation, phosphorylation, 0 acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl inositol) such that the homologue includes at least one epitope capable of eliciting an immune response against a flea peritrophin protein, and/or of binding to an antibody directed against a flea peritrophin protein. That is, when the N homologue is administered to an animal as an immunogen, using techniques known to S those skilled in the art, the animal will produce an immune response against at least one epitope of a natural flea peritrophin protein. The ability of a protein to effect an immune response can be measured using techniques known to those skilled in the art.

As used herein, the term "epitope" refers to the smallest portion of a protein or other antigen capable of selectively binding to the antigen binding site of an antibody or a T cell receptor. It is well accepted by those skilled in the art that the minimal size of a protein epitope is about four to six amino acids. As is appreciated by those skilled in the art, an epitope can include amino acids that naturally are contiguous to each other as well as amino acids that, due to the tertiary structure of the natural protein, are in sufficiently close proximity to form an epitope. According to the present invention, an epitope includes a portion of a protein comprising at least 4 amino acids, at least amino acids, at least 6 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids or at least 50 amino acids in length.

Flea peritrophin homologue proteins can be the result of natural allelic variation or natural mutation. Flea peritrophin protein homologues of the present invention can also be produced using techniques known in the art including, but not limited to, direct modifications to the protein or modifications to the gene encoding the protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis.

Flea peritrophin proteins of the present invention are encoded by flea peritrophin nucleic acid molecules. As used herein, flea peritrophin nucleic acid molecules include nucleic acid sequences related to natural flea peritrophin genes, and, preferably, to C. felis flea peritrophin genes. As used herein, flea peritrophin genes C include all regions such as regulatory regions that control production of flea peritrophin proteins encoded by such genes (such as, but not limited to, transcription, 00 translation or post-translation control regions) as well as the coding region itself, and any introns or non-translated coding regions. As used herein, a nucleic acid molecule that "includes" or "comprises" a sequence may include that sequence in one ec¢ contiguous array, or may include the sequence as fragmented exons such as is often found for a flea gene. As used herein, the term "coding region" refers to a continuous linear array of nucleotides that translates into a protein. A full-length coding region is that coding region that is translated into a full-length, a complete protein as would be initially translated in its natural millieu, prior to any post-translational modifications.

One embodiment of the present invention is a C. felis flea peritrophin gene that includes the nucleic acid sequence SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, 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:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and/or SEQ ID NO:49. These nucleic acid sequences are further described herein. For example, nucleic acid sequence SEQ ID NO: 1 represents the deduced sequence of the coding strand of a C. felis cDNA denoted herein as C.

felis peritrophin nucleic acid molecule nCfPL 1 09 6 the production of which is disclosed in the Examples. Nucleic acid molecule SEQ ID NO: 1 comprises an apparently full-length coding region. The complement of SEQ ID NO: 1 (represented herein by SEQ ID NO:3) refers to the nucleic acid sequence of the strand fully complementary to the strand having SEQ ID NO:1, which can easily be determined by.

those skilled in the art. Likewise, a nucleic acid sequence complement of any nucleic acid sequence of the present invention refers to the nucleic acid sequence of the nucleic acid strand that is fully complementary to can form a complete double

N

helix with) the strand for which the sequence is cited. It should be noted that since nucleic acid sequencing technology is not entirely error-free, SEQ ID NO: 1 (as well as 00 other nucleic acid and protein sequences presented herein) represents an apparent nucleic acid sequence of the nucleic acid molecule encoding a flea peritrophin protein of the present invention.

Translation of SEQ ID NO: 1, the coding strand of nCfPL 0 9 6 as well as Ctranslation of SEQ ID NO:4, the coding strand of nCfPL1,,l, which represents the coding region of nCfPL 109 yields a protein of 272 amino acids, denoted herein as PCfPL1 2 72 the amino acid sequence of which is presented in SEQ ID NO:2, assuming an initiation codon extending from nucleotide 6 to 8 of SEQ ID NO: 1, or from nucleotide 1 to nucleotide 3 of SEQ ID NO:4, respectively; and a stop codon extending from nucleotide 822 to 824 of SEQ ID NO: 1.

Translation of SEQ ID NO: 11, the coding strand of nCfPL2 1 46 s, as well as translation of SEQ ID NO: 14, the coding strand of nCfPL2 3 59 which represents the coding region of nCfPL2 4 6 yields a protein of 453 amino acids, denoted herein as PCfPL2 4 53 the amino acid sequence of which is presented in SEQ ID NO:12, assuming an initiation codon extending from nucleotide 3 to5 of SEQ ID NO: 11, or from nucleotide 1 to nucleotide 3 of SEQ ID NO:14, respectively; and a stop codon extending from nucleotide 1362 to 1364 of SEQ ID NO:11.

Translation of SEQ ID NO:16, the coding strand of nCfPL3 3 87 as well as translation of SEQ ID NO: 19, the coding strand of nCfPL3 2 43 which represents the coding region of nCfPL33,, yields a protein of 81 amino acids, denoted herein as PCfPL3 8 1 the amino acid sequence of which is presented in SEQ ID NO: 17, assuming an initiation codon extending from nucleotide 20-22 of SEQ ID NO: 16, or from nucleotide 1 to nucleotide 3 of SEQ ID NO:19, respectively; and a stop codon extending from nucleotide 263 to 265 of SEQ ID NO: 16.

Translation of SEQ ID NO:25, the coding strand of nCfPL4IA 1 0, as well as translation of SEQ ID NO:28, the coding strand of nCfPL4 8 which represents the coding region of nCfPL4i04, yields a protein of 285 amino acids, denoted herein as PCfPL4 2 85, the amino acid sequence of which is presented in SEQ ID NO:26, assuming an initiation codon extending from nucleotide 19-21 of SEQ ID NO:25, or from nucleotide 1 to nucleotide 3 of SEQ ID NO:28, respectively; and a stop codon extending from nucleotide 874 to 876 of SEQ ID 00Translation of SEQ ID NO:42, the coding strand of nCfPL532, as well as translation of SEQ ID NO:45, the coding strand of nCfPL5,, 91 which represents the coding region of nCfPL5,,1832, yields a protein of 397 amino acids, denoted herein as PCfPL 3 97 the amino acid sequence of which is presented in SEQ ID NO:43, assuming an initiation codon extending from nucleotide 146-148 of SEQ ID NO:42, or from nucleotide 1 to nucleotide 3 of SEQ ID NO:45, respectively; and a stop codon extending from nucleotide 1337 to 1339 of SEQ ID NO:42.

In one embodiment, a gene or other nucleic acid molecule of the present invention can be an allelic variant that includes a similar but not identical sequence to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ JID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQID NO:45, SEQ ID NO:46, SEQ ID NO:47, and/or SEQ ID NO:49. For example, an allelic variant of a C. felis peritrophin gene including SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQD NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID 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:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and/or SEQ ID NO:49 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:5, SEQ ID NO:6, -11- 0 SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID ^r NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID 0 NO:24, SEQ ID NO:25, 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:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID _r NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and/or SEQ ID NO:49, but which, due to natural variations caused by, for example, mutation or 0 recombination, has a similar but not identical sequence. Because natural selection 10 typically selects against alterations that affect function, allelic variants alleles corresponding to, or of, cited nucleic acid sequences) usually encode proteins having similar activity to that of the protein encoded by the gene to which they are being compared. Allelic variants of genes or nucleic acid molecules can also comprise alterations in the 5' or 3' untranslated regions of the gene in regulatory control regions), or can involve alternative splicing of a nascent transcript, thereby bringing alternative exons into juxtaposition. Allelic variants are well known to those skilled in the art and would be expected to occur naturally within a given flea species, since the genome is diploid, and sexual reproduction will result in the reassortment of alleles.

In one embodiment of the present invention, isolated flea peritrophin proteins are encoded by nucleic acid molecules that hybridize under stringent hybridization conditions to genes or other nucleic acid molecules encoding flea peritrophin proteins, respectively. The minimal size of flea peritrophinproteins of the present invention is a ns of theuresent size sufficient to be encoded by a nucleic acid molecule capable of forming a stable hybrid hybridizing under stringent hybridization conditions) with the complementary sequence of a nucleic acid molecule encoding the corresponding natural protein. The size of a nucleic acid molecule encoding such a protein is dependent on the nucleic acid composition and the percent homology between the flea peritrophin nucleic acid molecule and the complementary nucleic acid sequence. It can easily be understood that the extent of homology required to form a stable hybrid under stringent conditions can vary depending on whether the homologous sequences are interspersed throughout a given nucleic acid molecule or are clustered localized) in distinct regions on a given nucleic acid molecule.

Or The minimal size of a nucleic acid molecule capable of forming a stable hybrid 00 with a gene encoding a flea peritrophin protein is at least about 12 to about nucleotides in length if the nucleic acid molecule is GC-rich and at least about 15 to about 17 bases in length if it is AT-rich. The minimal size of a nucleic acid molecule used to encode a flea peritrophin protein homologue of the present invention is from N about 12 to about 18 nucleotides in length. Thus, the minimal size of flea peritrophin protein homologues of the present invention is from about 4 to about 6 amino acids in length. There is no limit, other than a practical limit, on the maximal size of a nucleic acid molecule encoding a flea peritrophin protein of the present invention because a nucleic acid molecule of the present invention can include a portion of a gene or cDNA or RNA, an entire gene or cDNA or RNA, or multiple genes or cDNA or RNA.

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.

Stringent hybridization conditions are determined based on defined physical properties of the flea peritrophin nucleic acid molecule to which the nucleic acid molecule is being hybridized, and can be defined mathematically. Stringent hybridization conditions are those experimental parameters that allow an individual skilled in the art to identify significant similarities between heterologous nucleic acid molecules. These conditions are well known to those skilled in the art. See, for example, Sambrook, et al., 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, and Meinkoth, et al., 1984, Anal. Biochem. 138, 267-284.

As explained in detail in the cited references, the determination of hybridization conditions involves the manipulation of a set of variables including the ionic strength in moles/liter), the hybridization temperature the concentration of nucleic acid helix destabilizing agents (such as formamide), the average length of the shortest hybrid duplex and the percent G C composition of the fragment to which an unknown nucleic acid molecule is being hybridized. For nucleic acid molecules of at least about 150 nucleotides, these variables are inserted into a standard mathematical formula to calculate the melting temperature, or Tm, of a given nucleic acid molecule.

As defined in the formula below, T is the temperature at which two complementary nucleic acid molecule strands will disassociate, assuming 100% complementarity 00 between the two strands: Ti= 81.5°C 16.6 log M 0.41(%G C) 500/n 0.61 (%formamide).

For nucleic acid molecules smaller than about 50 nucleotides, hybrid stability is

C")

defined by the dissociation temperature which is defined as the temperature at Swhich 50% of the duplexes dissociate. For these smaller molecules, the stability at a standard ionic strength is defined by the following equation: Td 4(G C) 2(A T).

A temperature of 5°C below Td is used to detect hybridization between perfectly matched molecules.

Also well known to those skilled in the art is how base pair mismatch, i.e.

differences between two nucleic acid molecules being compared, including noncomplementarity of bases at a given location, and gaps due to insertion or deletion of one or more bases at a given location on either of the nucleic acid molecules being compared, will affect Tm or Td for nucleic acid molecules of different sizes. For example, T m decreases about PC for each 1% of mismatched base pairs for hybrids greater than about 150 bp, and Td decreases about 5'C for each mismatched base pair for hybrids below about 50 bp. Conditions for hybrids between about 50 and about 150 base pairs can be determined empirically and without undue experimentation using standard laboratory procedures well known to those skilled in the art. These simple procedures allow one skilled in the art to set the hybridization conditions (by altering, for example, the salt concentration, the helix destabilizing compound concentration or the temperature) so that only nucleic acid hybrids with greater than a specified base pair mismatch will hybridize. Because one skilled in the art can easily determine whether a given nucleic acid molecule to be tested is less than or greater than about 50 nucleotides, and can therefore choose the appropriate formula for determining hybridization conditions, he or she can determine whether the nucleic acid molecule will hybridize with a given gene under conditions designed to allow a desired amount of base pair mismatch.

Hybridization reactions are often carried out by attaching the nucleic acid N, molecule to be hybridized to a solid support such as a membrane, and then hybridizing with a labeled nucleic acid molecule, typically referred to as a probe, suspended in a hybridization solution. Examples of common hybridization reaction techniques include, but are not limited to, the well-known Southern and northern blotting procedures. Typically, the actual hybridization reaction is done under non-stringent conditions, at a lower temperature and/or a higher salt concentration, and then high stringency is achieved by washing the membrane in a solution with a higher temperature and/or lower salt concentration in order to achieve the desired stringency.

For example, if the skilled artisan wished to identify a nucleic acid molecule that hybridizes under conditions that would allow less than or equal to 30% pair mismatch with a flea peritrophin nucleic acid molecule of about 150 bp in length or greater, the following conditions could preferably be used. The average G C content of flea DNA is about 37%, as calculated from known flea nucleic acid sequences. The unknown nucleic acid molecules would be attached to a support membrane, and the 150 bp probe would be labeled, e.g. with a radioactive tag. The hybridization reaction could be carried out in a solution comprising 2X SSC in the absence of nucleic acid helix destabilizing compounds, at a temperature of about 37°C (low stringency conditions). Solutions of differing concentrations of SSC can be made by one of skill in the art by diluting a stock solution of 20X SSC (175.3 gram NaCI and about 88.2 gram sodium citrate in 1 liter of water, pH 7) to obtain the desired concentration of SSC. The skilled artisan would calculate the washing conditions required to allow up to 30% base pair mismatch. For example, in a wash solution comprising 1X SSC in the absence of nucleic acid helix destabilizing compounds, the Tm of perfect hybrids would be about 77°C: 81.5 0 C 16.6 log (.15M) (0.41 x 73) (500/150) (0.61 x 0) 77.5 0

C.

Thus, to achieve hybridization with nucleic acid molecules having about 30% base pair mismatch, hybridization washes would be carried out at a temperature of less than or equal to 47.5°C. It is thus within the skill of one in the art to calculate additional hybridization temperatures based on the desired percentage base pair mismatch, formulae and G/C content disclosed herein. For example, it is appreciated by one skilled in the art that as the nucleic acid molecule to be tested for hybridization against nucleic acid molecules of the present invention having sequences specified herein becomes longer than 150 nucleotides, the Tm for a hybridization reaction allowing up 00 to 30% base pair mismatch will not vary significantly from 47.5 °C.

Furthermore, it is known in the art that there are commercially available computer programs for determining the degree of similarity between two nucleic acid or protein sequences. These computer programs include various known methods to determine the percentage identity and the number and length of gaps between hybrid nucleic acid molecules or proteins. Preferred methods to determine the percent identity among amino acid sequences and also among nucleic acid sequences include analysis using one or more of the commercially available computer programs designed to compare and analyze nucleic acid or amino acid sequences. These computer programs include, but are not limited to, the SeqLab® Wisconsin PackageTM Version 10.0-UNIX sequence analysis software, available from Genetics Computer Group, Madison, WI (hereinafter "SeqLab"); and DNAsis® sequence analysis software, version 2.0, available from Hitachi Software, San Bruno, CA (hereinafter "DNAsis").

Such software programs represent a collection of algorithms paired with a graphical user interface for using the algorithms. The DNAsis and SeqLab software, for example, employ a particular algorithm, the Needleman-Wunsch algorithm to perform pair-wise comparisons between two sequences to yield a percentage identity score, see Needleman, S.B. and Wunch, 1970, J. Mol. Biol., 48, 443. Such algorithms, including the Needleman-Wunsch algorithm, are commonly used by those skilled in the nucleic acid and amino acid sequencing art to compare sequences. A preferred method to determine percent identity among amino acid sequences and also among nucleic acid sequences includes using the Needleman-Wunsch algorithm, available in the SeqLab software, using the Pairwise Comparison/Gap function with the nwsgapdna.cmp scoring matrix, the gap creation penalty and the gap extension penalties set at default values, and the gap shift limits set at maximum (hereinafter referred to as "SeqLab default parameters"). An additional preferred method to determine percent identity among amino acid sequences and also among nucleic acid sequences includes using the Higgins-Sharp algorithm, available in the DNAsis -16o software, with the gap penalty set at 5, the number of top diagonals set at 5, the fixed N gap penalty set at 10, the k-tuple set at 2, the window size set at 5, and the floating gap penalty set at 10. A particularly preferred method to determine percent identity among 00 amino acid sequences and also among nucleic acid sequences includes using the Needleman-Wunsch algorithm available in the SeqLab software, using the SeqLab default parameters.

One embodiment of the present invention includes a flea peritrophin protein.

A preferred flea peritrophin protein includes a protein encoded by a nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to 30% base pair mismatch, preferably under conditions that allow less than or equal to base pair mismatch, preferably under conditions that allow less than or equal to base pair mismatch, preferably under conditions that allow less than or equal to 8% base pair mismatch, preferably under conditions that allow less than or equal to base pair mismatch or preferably under conditions that allow less than or equal to 2% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:46, and/or SEQ ID NO:49.

Another embodiment of the present invention includes a flea peritrophin protein encoded by a nucleic acid molecule that hybridizes under conditions comprising, hybridizing in a solution comprising IX SSC in the absence of nucleic acid helix destabilizing compounds, at a temperature of 37°C and washing in a solution comprising IX SSC in the absence of nucleic acid helix destabilizing compounds, at a temperature of 47°C, to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:46, and/or SEQ ID NO:49.

Another preferred flea peritrophin protein of the present invention includes a protein that is encoded by a nucleic acid molecule that is preferably at least -17- O identical, preferably at least 80% identical, preferably at least 90% identical, preferably at least 92% identical, preferably at least 95% identical or preferably at least 98% VQ identical to a nucleic acid molecule having the nucleic acid sequence SEQ ID NO: 1, 00SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:34, SEQ ID SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID N NO:42, SEQ ID NO:45, and/or SEQ ID NO:47; also preferred are fragments (i.e.

0 portions) of such proteins encoded by nucleic acid molecules that are at least N 10 nucleotides. Percent identity as used herein is determined using the Needleman- Wunsch algorithm, available in the SeqLab software using default parameters.

Additional preferred flea peritrophin proteins of the present invention include proteins having the amino acid sequence SEQ ID NO:2, SEQ ID NO: 12, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:40, SEQ ID NO:43, and/or SEQ ID NO:48, and proteins comprising homologues of a protein having the amino acid sequence SEQ ID NO:2, SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:40, SEQ ID NO:43, and/or SEQ ID NO:48, wherein such a homologue comprises at least one epitope that elicits an immune response against a protein having an amino acid sequence SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:40, SEQ ID NO:43, and/or SEQ ID NO:48. Likewise, also preferred are proteins encoded by nucleic acid molecules comprising nucleic acid sequence SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQID NO:11, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, and/or SEQ ID NO:47, or by homologues thereof.

A preferred isolated flea peritrophin protein of the present invention is a protein encoded by at least one of the following nucleic acid molecules: nCfPL ,196, nCfPL1,, 6 nCfPL2 5 nCfPL2, 2 79 nCfPL2 2 79 nCfPL2, 4 65 nCfPL2, 3 5 nCfPL3 3 7 nCfPL3 243 nCfPL4 960 nCfPL029, nCfP41, nCfPL4 855 nCfPL4 02 nCfPL5,, 3 8 nCfPL5n, 19 and/or nCfPL5 1 6 or allelic variants of any of these nucleic acid molecules. Also preferred is an isolated protein encoded by a nucleic acid V/1 molecule having nucleic acid sequence SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:6, 00 SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:28, SEQ ID SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, and/or SEQ 1 ID NO:47; or a protein encoded by an allelic variant of any of these listed nucleic acid molecules.

Preferred flea peritrophin proteins of the present invention include proteins having amino acid sequences that are at least 70%, preferably 80%, preferably preferably 92%, preferably 95%, preferably at least 98%, preferably at least 99%, or preferably 100% identical to amino acid sequence SEQ ID NO:2, SEQ ID NO: 12, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:40, SEQ ID NO:43, and/or SEQ ID NO:48; and proteins encoded by allelic variants of nucleic acid molecules encoding flea peritrophin proteins having amino acid sequences SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:40, SEQ ID NO:43, and/or SEQ ID NO:48. Also preferred are fragments thereof having at least amino acid residues.

In one embodiment of the present invention, C. felis peritrophin proteins comprise amino acid sequence SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:40, SEQ ID NO:43, and/or SEQ ID NO:48 (including, but not limited to, the proteins consisting of amino acid sequence SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:40, SEQ ID NO:43, and/or SEQ ID NO:48, fusion proteins and multivalent proteins), and proteins encoded by allelic variants of nucleic acid molecules encoding proteins having amino acid sequence SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:40, SEQ ID NO:43, and/or SEQ ID NO:48.

In one embodiment, a preferred flea peritrophin protein comprises an amino acid sequence of at least 6 amino acids, preferably at least 10 amino acids, preferably 0 at least 15 amino acids, preferably at least 20 amino acids, preferably at least 25 amino acids, preferably at least 30 amino acids, preferably at least 35 amino acids, preferably at least 40 amino acids, preferably at least 50 amino acids, preferably at least 75 amino 0 acids, preferably at least 100 amino acids, preferably at least 125 amino acids, preferably at least 150 amino acids, preferably at least 175 amino acids, preferably at least 200 amino acids, preferably at least 250 amino acids, preferably at least 300 Ir amino acids, preferably at least 350 amino acids, preferably at least 400 amino acids, or preferably at least 450 amino acids. In another embodiment, preferred flea O peritrophin proteins comprise full-length proteins, proteins encoded by full-length 10 coding regions, or post-translationally modified proteins thereof, such as mature proteins from which initiating methionine and/or signal sequences or "pro" sequences have been removed.

Additional preferred flea peritrophin proteins of the present invention include proteins encoded by nucleic acid molecules comprising at least a portion of nCfPLl 1 09 6 nCfPLls 16 nCfPL24 5 nCfPL2 1 2 nCfPL2 27 9, nCfPL2 1 46 nCfPL2 1 3 59 nCfPL3 3 87 nCfPL3 243 nCfPL4 960 nCfPL4 1 029 nCfPI 8, nCfPL 5 nCfPL4 8 5 3 nCfPL5 1 8 32 nCfPL5,, 9 and/or nCfPL5 1 as well as flea peritrophin proteins encoded by allelic variants of such nucleic acid molecules. A portion of such flea peritrophin nucleic acid molecule is preferably at least 25 nucleotides in length.

Also preferred are flea peritrophin proteins encoded by nucleic acid molecules having nucleic acid sequences comprising at least a portion of SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:34, SEQ ID SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, and/or SEQ ID NO:47, as well as allelic variants of these nucleic acid molecules. A portion of such flea peritrophin nucleic acid molecule is preferably at least 25 nucleotides in length.

In another embodiment, a preferred flea peritrophin protein of the present invention is encoded by a nucleic acid molecule comprising at least 20 nucleotides, preferably at least 25 nucleotides, preferably at least 30 nucleotides, preferably at least 0 40 nucleotides, preferably at least 50 nucleotides, preferably at least 75 nucleotides, preferably at least 100 nucleotides, preferably at least 125 nucleotides, preferably at C/ least 150 nucleotides, preferably at least 175 nucleotides, preferably at least 200 00 nucleotides, preferably at least 250 nucleotides, preferably at least 350 nucleotides, preferably at least 450 nucleotides, preferably at least 550 nucleotides, preferably at Sleast 650 nucleotides, preferably at least 750 nucleotides, preferably at least 1000 Snucleotides, preferably at least 1100 nucleotides, preferably at least 1200 nucleotides, preferably at least 1500 nucleotides, preferably at least 1750 nucleotides, or preferably Sat least 1850 nucleotides in length. Within this embodiment is a flea peritrophin N 10 protein encoded by at least a portion of nCfPL 1 0 o 9 6 nCfPL1 816 nCfPL244, nCfPL2 1 279 nCfPL2 2 79 nCfPL2 1 4 65 nCfPL2 1359 nCfPL3 3 8 7 nCfPL3 2 43 nCfPL4 9 6 nCfPL402 9 nCfPL4A148, nCfPL4 8 55 nCfPL4A2, nCfPL5 1 5 3 nCfPL5 1 3 2 nCfPL5 1 1 91 and/or 1 6 1 or by an allelic variant of any of these nucleic acid molecules. Preferred flea peritrophin proteins of the present invention are encoded by nucleic acid molecules comprising apparently full-length flea peritrophin coding region, i.e., nucleic acid molecules encoding an apparently full-length flea peritrophin protein.

Preferred flea peritrophin proteins of the present invention can be used to develop inhibitors that, when administered to an animal in an effective manner, are capable of protecting that animal from flea infestation. In accordance with the present invention, the ability of an inhibitor of the present invention to protect an animal from flea infestation refers to the ability of that protein to, for example, treat, ameliorate and/or prevent infestation caused by fleas. In particular, the phrase "to protect an animal from flea infestation" refers to reducing the potential for flea population expansion on and around the animal reducing the flea burden). Preferably, the flea population size is decreased, optimally to an extent that the animal is no longer bothered by fleas. A host animal, as used herein, is an animal from which fleas can feed by attaching to and feeding through the skin of the animal. Fleas, and other ectoparasites, can live on a host animal for an extended period of time or can attach temporarily to an animal in order to feed. At any given time, a certain percentage of a flea population can be on a host animal whereas the remainder can be in the environment of the animal. Such an environment can include not only adult fleas, but o also flea eggs and/or flea larvae. The environment can be of any size such that fleas in the environment are able to jump onto and off of a host animal. For example, the environment of an animal can include plants, such as crops, from which fleas infest an 00 animal. As such, it is desirable not only to reduce the flea burden on an animal per se, but also to reduce the flea burden in the environment of the animal.

SSuitable fleas to target include any flea that is essentially incapable of causing disease in an animal administered an inhibitor of the present invention. As such, fleas to target include any flea that produces a protein that can be targeted by an inhibitory compound that inhibits a flea flea peritrophin protein function, thereby resulting in the decreased ability of the parasite to cause disease in an animal. Preferred fleas to target include fleas of the following genera: Ctenocephalides, Cyopsyllus, Diamanus (Oropsylla), Echidnophaga, Nosopsyllus, Pulex, Tunga, and Xenopsylla, with those of the species Ctenocephalides canis, Ctenocephalidesfelis, Diamanus montanus, Echidnophaga gallinacea, Nosopsyllusfaciatus, Pulex irritans, Pulex simulans, Tunga penetrans and Xenopsylla cheopis being more preferred, with C. felis being even more preferred. Such fleas are also preferred for the isolation of proteins or nucleic acid molecules of the present invention.

One embodiment of a flea peritrophin protein of the present invention is a fusion protein that includes a flea peritrophin 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; and/or assist in purification of a flea peritrophin protein by affinity chromatography). A suitable fusion segment can be a domain of any size that has the desired function imparts increased stability, imparts increased immunogenicity to a protein, and/or simplifies purification of a protein).

Fusion segments can be joined to amino and/or carboxyl termini of the flea peritrophin-containing domain of the protein and can be susceptible to cleavage in order to enable straight-forward recovery of a flea peritrophin 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 a flea peritrophin-containing domain.

O Preferred fusion segments include a metal binding domain a poly-histidine segment); an immunoglobulin binding domain Protein A; Protein G; T cell; B cell; Fc receptor or complement protein antibody-binding domains); a sugar binding 00 domain a maltose binding domain); and/or a "tag" domain at least a portion of P-galactosidase, a strep tag peptide, a T7 tag peptide, a FlagTM peptide, or other Sdomains that can be purified using compounds that bind to the domain, such as r> monoclonal antibodies). More preferred fusion segments include metal binding domains, such as a poly-histidine segment; a maltose binding domain; a strep tag r peptide, such as that available from Biometra in Tampa, FL; and an S10 peptide.

The present invention also includes mimetopes of flea peritrophin proteins of the present invention. As used herein, a mimetope of a flea peritrophin protein of the present invention refers to any compound that is able to mimic the activity of such a flea peritrophin protein, often because the mimetope has a structure that mimics the particular flea peritrophin protein. Mimetopes can be, but are not limited to: peptides that have been modified to decrease their susceptibility to degradation such as all-D retro peptides; anti-idiotypic and/or catalytic antibodies, or fragments thereof; nonproteinaceous immunogenic portions of an isolated protein 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.

Another embodiment of the present invention is an isolated nucleic acid molecule comprising a flea peritrophin nucleic acid molecule, i.e. a nucleic acid molecule that can be isolated from a flea cDNA library. As used herein, flea peritrophin nucleic acid molecules has the same meaning as flea peritrophin nucleic acid molecule. The identifying characteristics of such nucleic acid molecules are heretofore described. A nucleic acid molecule of the present invention can include an isolated natural flea peritrophin gene or a homologue thereof, the latter of which is described in more detail below. A nucleic acid molecule of the present invention can include one or more regulatory regions, full-length or partial coding regions, or N "combinations thereof. The minimal size of a nucleic acid molecule of the present invention is a size sufficient to allow the formation of a stable hybrid 0 hybridization under stringent hybridization conditions) with the complementary sequence of another nucleic acid molecule. As such, the minimal size of a flea peritrophin nucleic acid molecule of the present invention is from 12 to 18 nucleotides in length.

N, In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu that has been 10 subjected to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA. As such, "isolated" does not reflect the extent to which the nucleic acid molecule has been purified. Isolated flea peritrophin nucleic acid molecules of the present invention, or homologues thereof, can be isolated from a natural source or produced using recombinant DNA technology polymerase chain reaction (PCR) amplification or cloning) or chemical synthesis. Isolated flea peritrophin nucleic acid molecules, and homologues thereof, can include, for example, natural allelic variants and nucleic acid molecules modified by nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode a flea peritrophin protein of the present invention.

A flea peritrophin nucleic acid molecule homologue can be produced using a number of methods known to those skilled in the art, see, for example, Sambrook et al., ibid. For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis and recombinant DNA techniques such as site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments, PCR amplification, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules, and combinations thereof. Nucleic acid molecule homologues can be selected by hybridization with flea peritrophin nucleic acid molecules or by screening the function of a protein encoded by the nucleic acid molecule ability to elicit an immune response against at least one epitope of a flea peritrophin protein, to selectively bind to an antibody that binds a flea peritrophin protein or to effect flea peritrophin activity).

An isolated flea peritrophin nucleic acid molecule of the present invention can Sinclude a nucleic acid sequence that encodes at least one flea peritrophin protein of the present invention respectively, examples of such proteins being disclosed herein.

SAlthough the phrase "nucleic acid molecule" primarily refers to the physical nucleic r acid molecule and the phrase "nucleic acid sequence" primarily refers to the sequence Sof 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 10 sequence, being capable of encoding a flea peritrophin protein.

A preferred nucleic acid molecule of the present invention, when administered to an animal, is capable of protecting that animal from flea infestation. As will be disclosed in more detail below, a nucleic acid molecule of the present invention can be, or encode, an antisense RNA, a molecule capable of triple helix formation, a ribozyme, or other nucleic acid-based drug compound. In additional embodiments, a nucleic acid molecule of the present invention can encode a protective protein a flea peritrophin protein of the present invention), the nucleic acid molecule being delivered to the animal, for example, by direct injection as a genetic vaccine) or in a vehicle such as a recombinant virus vaccine or a recombinant cell vaccine.

In one embodiment of the present invention, a preferred flea peritrophin nucleic acid molecule includes an isolated nucleic acid molecule that hybridizes under conditions that preferably allow less than or equal to 30% base pair mismatch, preferably under conditions that allow less than or equal to 20% base pair mismatch, preferably under conditions that allow less than or equal to 10% base pair mismatch preferably under conditions that allow less than or equal to 5% base pair mismatch or preferably under conditions that allow less than or equal to 2% base pair mismatch with a nucleic acid molecule selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID SNO: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:37, SEQ ID Vry NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID 00 NO:45, SEQ ID NO:46, SEQ ID NO:47, and/or SEQ ID NO:49.

Another embodiment of the present invention includes a flea peritrophin Snucleic acid molecule, wherein said nucleic acid molecule hybridizes under conditions r comprising, hybridizing in solution comprising 1X SSC in the absence of nucleic acid helix destabilizing compounds, at a temperature of 37°C and washing in a 0 solution comprising IX SSC in the absence of nucleic acid helix destabilizing compounds, at a temperature of 47 0 C, to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, 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:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and/or SEQ ID NO:49. Additional preferred nucleic acid molecules of the present invention include oligonucleotides of an isolated nucleic acid molecule, wherein said nucleic acid molecule hybridizes under conditions comprising, (a) hybridizing in solution comprising 1X SSC in the absence of nucleic acid helix destabilizing compounds, at a temperature of 37 0 C and washing in a solution comprising lX SSC in the absence of nucleic acid helix destabilizing compounds, at a temperature of 47 0 C, to an isolated nucleic acid molecule selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, 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 -26- SEQ IDNO:36, SEQ ID NO:37, SEQ ED NO:38, SEQ ID NO:39, SEQID NO:41, (N SEQ DD NO:42, SEQ H) NO:44, SEQ BD NO:45, SEQ ID NO:46, SEQ DD NO:47, and/or SEQ D NO:49, wherein said oligonucleotide comprises at least 25 nucleotides.

00 Additional preferred flea peritrophin nucleic acid molecules of the present invention include nucleic acid molecules comprising a nucleic. acid sequence that is preferably at least 70%, preferably at least 80%, preferably at least 90%, preferably at least 92%, preferably at least 95%, or preferably at least 98% identical to a nucleic acid sequence selected from the group consisting of SEQ I1D NO:l1, SEQ 1D NO:3, SEQ ED NO:4, SEQ ID NO:5, SEQ IID NO:6, SEQID NO:7, SEQ ED NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IID NO: 13, SEQ ID NO: 14, SEQ ID NO: SE DN:1,SQI O:1,SQE O 9 E I O2,SQI O 1 SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:32, SEQ ID NO:27, SEQ DD NO:28, SEQ ID NO:29, SEQ DD NO:30, SEQ IG) NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ JID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:46, SEQ IID NO:47, and/or SEQ ID NO:49. Also preferred are oligonucleotides of any of such nucleic acid molecules. Percent identity as used herein is determined using the Needleman-Wunsch algorithm, available in the SeqLab software using default parameters.

One embodiment of the present invention is a nucleic acid molecule comprising all or part of nucleic acid molecules nCfPL 1096 nCfPL 81 6 nCfPL244,, nCfPL2 279 nCfPL2 279 nCfPL2 1465 nCfPL2 1 3 5 9 nCfPL3 3 8 7 nCfPL3 243 nCfPA 960 nCfPM 1029 nCfPL-4 1 04 8 nCfPL4 855 nCfPLAscu, nCfPL5 1513' nCfPL5 1832 nCfPL5 1 1 9 and/or nCfPL5 11 61 or allelic. variants of these nucleic acid molecules. Another preferred nucleic acid molecule of the present invention includes at least a portion of nucleic acid sequence SEQ ID NO: 1, SEQ ED NO: 3, SEQ ID NO:4, SEQ IID NO: SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO: 8, SEQ ID NO:9, SEQ ED NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQIED NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 18, SEQID NO: 19, SEQ DNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IID NO:30, SEQ ID NO:32, SEQ IID NO:33, SEQ ID NO:34, SEQ ID -27- SEQ ID NO:36, SEQ ID NO:37, SEQ ID N0:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID SNO:47, and/or SEQ ID NO:49, as well as allelic variants of nucleic acid molecules 00 having these nucleic acid sequences and homologues of nucleic acid molecules having these nucleic acid sequences; preferably such a homologue encodes or is complementary to a nucleic acid molecule that encodes at least one epitope that elicits t an immune response against a protein having an amino acid sequence SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO: 17, SEQ ID NO:26, SEQ ID NO:31, SEQ ID SEQ ID NO:43, and/or SEQ ID NO:48. 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.

In one embodiment, a flea peritrophin nucleic acid molecule of the present invention encodes a protein having an amino acid sequence that is at least preferably at least 80%, preferably at least 90%, preferably at least 95%, preferably at least 98%, preferably at least 99%, or preferably at least 100% identical to SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:31, SEQ ID SEQ ID NO:43, and/or SEQ ID NO:48. The present invention also includes a flea peritrophin nucleic acid molecule encoding a protein having at least a portion of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:40, SEQ ID NO:43, and/or SEQ ID NO:48, as well as allelic variants of a nucleic acid molecule encoding a protein having these sequences, including nucleic acid molecules that have been modified to accommodate codon usage properties of the cells in which such nucleic acid molecules are to be expressed.

In another embodiment, a preferred flea peritrophin nucleic acid molecule of the present invention comprises a nucleic acid molecule comprising at least nucleotides, preferably at least 25 nucleotides, preferably at least 30 nucleotides, preferably at least 40 nucleotides, preferably at least 50 nucleotides, preferably at least 75 nucleotides, preferably at least 100 nucleotides, preferably at least 125 nucleotides, preferably at least 150 nucleotides, preferably at least 175 nucleotides, preferably at Sleast 200 nucleotides, preferably at least 250 nucleotides, preferably at least 350 Snucleotides, preferably at least 450 nucleotides, preferably at least 550 nucleotides, Spreferably at least 650 nucleotides, preferably at least 750 nucleotides, preferably at least 1000 nucleotides, preferably at least 1100 nucleotides, preferably at least 1200 least 1000 nucleotides, preferably at least 15 00 nucleotides, preferably at least 1750 nucleotides, nucleotides, preferably at least 1500 nucleotides, preferably at least 1750 nucleotides, or preferably at least 1850 nucleotides in length.

In another embodiment, a preferred flea peritrophin nucleic acid molecule Sencodes a protein comprising at least 6 amino acids, preferably at least 10 amino acids, preferably at least 20 amino acids, preferably at least 30 amino acids, preferably at least 40 amino acids, preferably at least 50 amino acids, preferably at least 75 amino acids, preferably at least 100 amino acids, preferably at least 200 amino acids, preferably at least 300 amino acids, preferably at least 400 amino acids, or preferably at least 450 amino acids.

In another embodiment, a preferred flea peritrophin nucleic acid molecule of the present invention comprises an apparently full-length flea peritrophin coding region, the preferred nucleic acid molecule encodes an apparently full-length flea peritrophin protein, respectively, or a post-translationally modified protein thereof. In one embodiment, a preferred flea peritrophin nucleic acid molecule of the present invention encodes a mature protein.

In another embodiment, a preferred flea peritrophin nucleic acid molecule of the present invention comprises a nucleic acid molecule comprising SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID 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:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and/or SEQ ID NO:49, or a fragment thereof.

-29- SA fragment of a flea peritrophin nucleic acid molecule of the present invention a preferably comprises at least 15 nucleotides, preferably at least 18 nucleotides, r preferably at least 21 nucleotides, preferably at least 25 nucleotides, preferably at least 00 30 nucleotides, preferably at least 35 nucleotides, preferably at least 40 nucleotides, preferably at least 50 nucleotides, preferably at least 75 nucleotides, preferably at least 100 nucleotides, preferably at least 125 nucleotides, preferably at least 150 nucleotides, preferably at least 175 nucleotides, preferably at least 200 nucleotides, preferably at least 250 nucleotides, preferably at least 350 nucleotides, preferably at least 450 nucleotides, preferably at least 550 nucleotides, preferably at least 650 10 nucleotides, preferably at least 750 nucleotides, preferably at least 1000 nucleotides, preferably at least 1100 nucleotides, preferably at least 1200 nucleotides, preferably at least 1500 nucleotides, preferably at least 1750 nucleotides, or preferably at least 1850 nucleotides identical in sequence to a corresponding contiguous sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, 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:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and/or SEQ ID NO:49.

The phrase, a nucleic acid molecule comprising at least contiguous, or consecutive nucleotides identical in sequence to at least contiguous, or consecutive nucleotides of a nucleic acid molecule selected from the group consisting of SEQ ID refers to an "x"-nucleotide in length nucleic acid molecule that is identical in sequence to an "x"-nucleotide portion of SEQ ID as well as to nucleic acid molecules that are longer in length than The additional length may be in the form of nucleotides that extend from either the 5' or the 3' end(s) of the contiguous identical "x"-nucleotide portion. The 5' and/or 3' extensions can include one or more extensions that have no identity to a molecule of the present invention, as well as extensions that Sshow similarity or identity to cited nucleic acids sequences or portions thereof.

SKnowing the nucleic acid sequences of certain flea peritrophin nucleic acid 00 molecules of the present invention allows one skilled in the art to, for example, (a) make copies of those nucleic acid molecules, obtain nucleic acid molecules including at least a portion of such nucleic acid molecules nucleic acid molecules including full-length genes, full-length coding regions, regulatory control sequences, truncated coding regions), and obtain other flea peritrophin nucleic acid molecules.

Such nucleic acid molecules can be obtained in a variety of ways including screening appropriate expression libraries with antibodies of the present invention; traditional cloning techniques using oligonucleotide probes of the present invention to screen appropriate libraries; and PCR amplification of appropriate libraries or DNA using oligonucleotide primers of the present invention. Preferred libraries to screen or from which to amplify nucleic acid molecules include cDNA libraries as well as genomic DNA libraries. Similarly, preferred DNA sources to screen or from which to amplify nucleic acid molecules include cDNA and genomic DNA. Techniques to clone and amplify genes are disclosed, for example, in Sambrook et al., ibid.

The present invention also includes nucleic acid molecules that are oligonucleotides capable of hybridizing, under stringent hybridization conditions, with complementary regions of other, preferably longer, nucleic acid molecules of the present invention such as those comprising C. felis peritrophin nucleic acid molecules or other flea peritrophin nucleic acid molecules. Oligonucleotides of the present invention can be RNA, DNA, or derivatives of either. The minimum size of such oligonucleotides is the size required for formation of a stable hybrid between an oligonucleotide and a complementary sequence on a nucleic acid molecule of the present invention. A preferred oligonucleotide of the present invention has a maximum size of preferably 100 to 200 nucleotides. The present invention includes oligonucleotides that can be used as, for example, probes to identify nucleic acid molecules, primers to produce nucleic acid molecules, or therapeutic reagents to inhibit flea peritrophin protein production or activity as antisense-, triplex formation-, ribozyme- and/or RNA drug-based reagents). The present invention also -31- 0 includes the use of such oligonucleotides to protect animals from disease using one or e. more of such technologies. Appropriate oligonucleotide-containing therapeutic compositions can be administered to an animal using techniques known to those 00 skilled in the art.

One embodiment of the present invention includes a recombinant vector, which includes at least one isolated nucleic acid molecule of the present invention, inserted r into any vector capable of delivering the nucleic acid molecule into a host cell. Such a rvector contains heterologous nucleic acid sequences, that is nucleic acid sequences that are not naturally found adjacent to nucleic acid molecules of the present invention and that preferably are derived from a species other than the species from which the nucleic acid molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid. Recombinant vectors can be used in the cloning, sequencing, and/or otherwise manipulating of flea peritrophin nucleic acid molecules of the present invention.

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 direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, parasite, insect, other animal, and plant cells. Preferred expression vectors of the present invention can direct gene expression in bacterial, yeast, insect and mammalian cells, and more preferably in the cell types disclosed herein.

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 -32- O recombinant cell and that control the expression of nucleic acid molecules of the Spresent invention. In particular, recombinant molecules of the present invention include transcription control sequences. Transcription control sequences are 0 sequences that control the initiation, elongation, and termination of transcription.

Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences.

Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety Sof such transcription control sequences are known to those skilled in the art. Preferred 10 transcription control sequences include those that function in bacterial, yeast, or insect and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda (such as lambda PL and lambda pR and fusions that include such promoters), bacteriophage T7, T7lac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoter, antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as immediate early promoter), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells. Additional suitable transcription control sequences include tissue-specific promoters and enhancers as well as lymphokineinducible promoters promoters inducible by interferons or interleukins).

Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with fleas, such as C.

felis transcription control sequences.

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 nCfPLI 1 0 9 6 nCfPL1 8 1 6 nCfPL24 5 nCfPL2 1 279 nCfPL2 2 9 nCfPL2 1465 nCfPL2 35 nCfPL3 38 7 nCfPL3 243 nCfPL490, nCfPLA 1 029 nCfPL40 8 nCfPL4 85 5 nCfPL4 8 02 1 51 3 nCfPL5 1 3 2 nCfPL51191, and/or nCfPL5 1 1 11 1191' n/rnf O Recombinant molecules of the present invention may also contain secretory signals signal segment nucleic acid sequences) to enable an expressed flea r) peritrophin protein of the present invention to be secreted from the cell that produces 0 the protein and/or contain fusion sequences which lead to the expression of nucleic acid molecules of the present invention as fusion proteins. Examples of suitable signal Ssegments include any signal segment capable of directing the secretion of a protein of Sthe present invention. Preferred signal segments include, but are not limited to, tissue C plasminogen activator interferon, interleukin, growth hormone, 0 histocompatibility and viral envelope glycoprotein signal segments. Suitable fusion C' 10 segments encoded by fusion segment nucleic acids are disclosed herein. In addition, a nucleic acid molecule of the present invention can be joined to a fusion segment that directs the encoded protein to the proteosome, such as a ubiquitin fusion segment.

Eukaryotic recombinant molecules may also include intervening and/or untranslated sequences surrounding and/or within the nucleic acid sequences of nucleic acid molecules of the present invention.

Another embodiment of the present invention includes a recombinant cell comprising a host cell transformed with one or more recombinant molecules of the present invention. Transformation of a nucleic acid molecule into a cell can be accomplished by any method by which a nucleic acid molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. It is to be noted that a cell line refers to any recombinant cell of the present invention that is not a transgenic animal. Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed 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 flea peritrophin nucleic acid molecules disclosed herein. Preferred nucleic acid molecules with which to transform a cell include nCfPL 1 ,96, nCfPL1 816 nCfPL244 5 nCfPL2 2 79 nCfPL2 27 9 nCfPL2 46 O nCfPL2 1 3 9 nCfPL3 38 7 nCfPL3 243 nCfPL 960 nCfPL4 1029 nCfPLA48, nCfP nCfPL4so2, nCfPL5 51 3 nCfPL5, 32 nCfPL5,,,, and/or nCfPL5.

1 Suitable host cells to transform include any cell that can be transformed with a 00 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 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 naturally) capable of producing flea peritrophin proteins of the present invention or can be capable of producing such proteins after being transformed with at least one nucleic acid molecule of the present invention. Host cells of the present invention can be any cell capable of producing at least one protein of the present invention, and include bacterial, fungal (including yeast), parasite (including helminth, protozoa and ectoparasite), other insect, other animal and plant cells. Preferred host cells include bacterial, mycobacterial, yeast, insect and mammalian cells. More preferred host cells include Salmonella, Escherichia, Bacillus, Caulobacter, Listeria, Saccharomyces, Pichia, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells (Madin- Darby canine kidney cell line), CRFK cells (Crandell feline kidney cell line), CV-1 cells (African monkey kidney cell line used, for example, to culture raccoon poxvirus), COS 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 .3987 and SR-11 4072; Caulobacter; Pichia; Spodoptera frugiperda; Trichoplusia ni; BHK cells; MDCK cells; CRFK cells; CV-1 cells; COS cells; Vero cells; and non-tumorigenic mouse myoblast G8 cells ATCC CRL 1246). Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK 31 cells and/or HeLa cells. In one embodiment, the proteins may be expressed as heterologous proteins in myeloma cell lines employing immunoglobulin promoters.

A recombinant cell is preferably produced by transforming a host cell with one N. 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 00 more transcription control sequences, examples of which are disclosed herein. The phrase operatively linked refers to insertion of a nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when C€3 transformed into a host cell.

rA recombinant cell of the present invention includes any cell transformed with at least one of any nucleic acid molecule of the present invention. Suitable and preferred nucleic acid molecules as well as suitable and preferred recombinant molecules with which to transfer cells are disclosed herein.

Recombinant cells of the present invention can also be co-transformed with one or more recombinant molecules including flea peritrophin 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 to produce multivalent vaccines).

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 promoters, operators, enhancers), substitutions or modifications of translational control signals 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 o production during fermentation. The activity of an expressed recombinant protein of Sthe present invention may be improved by fragmenting, modifying, or derivatizing nucleic acid molecules encoding such a protein.

00 Isolated flea peritrophin 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 N cell capable of expressing the protein under conditions effective to produce the protein, and recovering the protein. A preferred cell to culture is a recombinant cell of the present invention. Effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. An effective, medium refers to any medium in which a cell is cultured to produce a flea peritrophin 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.

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, 0 chromatofocusing and differential solubilization. Proteins of the present invention are preferably retrieved in "substantially pure" form. As used herein, "substantially pure" Srefers to a purity that allows for the effective use of the protein as a therapeutic 0 composition or diagnostic. A therapeutic composition for animals, for example, should exhibit no substantial toxicity and preferably should be capable of stimulating Sthe production of antibodies in a treated animal.

The present invention also includes isolated removed from their natural Smilieu) antibodies that selectively bind to a flea peritrophin protein of the present invention or a mimetope thereof anti-flea peritrophin antibodies). As used herein, the term "selectively binds to" a 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 ELISA), immunoblot assays, etc.; see, for example, Sambrook et al., ibid., and Harlow, et al., 1988, Antibodies, a Laboratoy Manual, Cold Spring Harbor Labs Press; Harlow et al., ibid. An anti-flea peritrophin antibody of the present invention preferably selectively binds to a flea peritrophin protein, respectively, in such a way as to inhibit the function of that protein.

Isolated antibodies of the present invention can include antibodies in serum, or antibodies that have been purified to varying degrees. Antibodies of the present invention can be polyclonal or monoclonal, or can be functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies or chimeric antibodies that can bind to one or more epitopes.

A preferred method to produce antibodies of the present invention includes administering to an animal an effective amount of a protein, peptide or mimetope thereof of the present invention to produce the antibodies and recovering the antibodies. In another method, antibodies of the present invention are produced recombinantly using techniques as heretofore disclosed to produce flea peritrophin 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.

Jo- O 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 S(a) as therapeutic compounds to passively immunize an animal in order to protect the 0 animal from fleas susceptible to treatment by such antibodies and/or as tools to screen expression libraries and/or to recover desired proteins of the present invention Sfrom a mixture of proteins and other contaminants. Furthermore, antibodies of the present invention can be used to target cytotoxic agents to fleas in order to directly kill such fleas. Targeting can be accomplished by conjugating 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.

One embodiment of the present invention is a therapeutic composition that, when administered to an animal susceptible to flea infestation, is capable of protecting that animal from flea infestation. Therapeutic compositions of the present invention include at least one of the following protective molecules: an isolated flea peritrophin protein; a mimetope of an isolated flea peritrophin protein; an isolated flea peritrophin nucleic acid molecule; and/or a compound derived from said isolated flea peritrophin protein that inhibits flea peritrophin protein activity. A therapeutic composition of the present invention can further comprise a component selected from the group of an excipient, a carrier, and/or an adjuvant; these components are described further herein.

As used herein, a protective molecule or protective compound refers to a compound that, when administered to an animal in an effective manner, is able to treat, ameliorate, and/or prevent flea infestation. Preferred fleas to target are heretofore disclosed. One example of a protective molecule is a vaccine, such as, but not limited to, a naked nucleic acid vaccine, a recombinant virus vaccine, a recombinant cell vaccine, and a recombinant protein vaccine. Another example of a protective molecule is a compound that inhibits flea peritrophin protein activity, such as an isolated antibody that selectively binds to a flea peritrophin protein, a substrate analog of a flea peritrophin protein, anti-sense-, triplex formation-, ribozyme-, and/or RNA drug-based compounds, or other inorganic or organic molecules that inhibit flea peritrophin protein activity. Inhibiting flea peritrophin protein activity can refer to the ability of a compound to reduce the activity of flea peritrophin proteins. Inhibiting flea O peritrophin protein activity can also refer to the ability of a compound to reduce the Samount of flea peritrophin protein in a flea.

/I One embodiment of the present invention is a therapeutic composition 0 comprising an excipient and a compound selected from the group consisting of: an isolated nucleic acid molecule selected from the group consisting of a flea cDNA molecule and a flea RNA molecule, wherein said nucleic acid molecule is selected from the group consisting of a nucleic acid molecule at least 25 nucleotides in length that hybridizes with a nucleic acid molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:4, 10 SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32, and a nucleic acid molecule at least 35 nucleotides in length that hybridizes with a nucleic acid molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID SEQ ID NO:46, SEQ ID NO:47, and SEQ D NO:49; wherein said hybridization of (a) and is performed under conditions comprising hybridizing in a solution comprising 1X SSC in the absence of nucleic acid helix destabilizing compounds, at a temperature of 37°C and washing in a solution comprising 1X SSC in the absence of helix destabilizing compounds, at a temperature of 47 0 C; an isolated protein selected from the group consisting of a protein encoded by a nucleic acid molecule selected from the group consisting of a nucleic acid molecule at least 25 nucleotides in length that hybridizes with a nucleic acid molecule having 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:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID SEQ ID NO: 11, SEQ ID NO:13, SEQ ID NO:14, SEQ ED NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID SNO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32, and (ii) a nucleic acid molecule at least 35 nucleotides in length that hybridizes with a nucleic acid molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NO:33, 0 SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID SSEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:49; wherein said hybridization of (i) and (ii) is performed under conditions comprising hybridizing in a solution comprising IX SSC in the absence of nucleic acid helix destabilizing compounds, at a 0 temperature of 37 0 C and washing in a solution comprising 1X SSC in the absence of helix destabilizing compounds, at a temperature of 47 0 C; and an isolated protein, comprising at least 10 amino acids identical in sequence to a 10 amino acid portion of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:40, SEQ ID NO:43, and SEQ ID NO:48; and an isolated antibody that selectively binds to a protein as set forth in Another embodiment of the present invention includes a method to reduce flea infestation in an animal susceptible to flea infestation. Such a method includes the step of administering to the animal a therapeutic molecule comprising a protective compound selected from the group consisting of an isolated flea peritrophin protein; a mimetope of an isolated flea peritrophin protein; an isolated flea peritrophin nucleic acid molecule; and a compound derived from an isolated flea peritrophin protein that inhibits flea peritrophin protein activity.

Therapeutic compositions of the present invention can be administered to any animal susceptible to flea infestation, preferably to mammals, and more preferably to dogs, cats, humans, ferrets, horses, cattle, sheep, and other pets, economic food animals, work animals and/or zoo animals. Preferred animals to protect against flea infestation include dogs, cats, humans, and ferrets, with dogs and cats being particularly preferred.

As used herein, the term derived, or the term derived from, refers to a peptide, antibody, mimetope, nucleic acid molecule, or other compound that was obtained from or using a flea peritrophin protein or nucleic acid molecule of the present invention.

-41- O Methods to obtain derivatives from a flea peritrophin molecule of the present invention are known in the art, and as such include, but are not limited to molecular modeling of flea peritrophin proteins to determine active sites, and predicting from these active 00 sites smaller fragments and/or mimetopes that retain and/or mimic these active sites, thereby inhibiting flea peritrophin protein activity. Other inhibitors of flea peritrophin activity can also be obtained in a variety of ways, including but not limited to Cc€ screening of peptide or small chemical compound libraries against flea peritrophin N proteins of the present invention; and screening of polyclonal or monoclonal antibodies to find antibodies that specifically bind flea peritrophin proteins of the present invention.

A flea peritrophin protein inhibitor of the present invention an inhibitor of a flea peritrophin protein) is identified by its ability to mimic, bind to, modify, or otherwise interact with, a flea peritrophin protein, thereby inhibiting the activity of a natural flea peritrophin protein. Suitable inhibitors of flea peritrophin protein activity are compounds that inhibit flea peritrophin protein activity in at least one of a variety of ways: by binding to or otherwise interacting with or otherwise modifying flea peritrophin protein sites; by binding to the flea peritrophin protein and thus reducing the availability of the flea peritrophin protein in solution; by mimicking a flea peritrophin protein; and by interacting with other regions of the flea peritrophin protein to inhibit flea peritrophin protein activity, for example, by allosteric interaction.

Flea peritrophin protein inhibitors can be used directly as compounds in compositions of the present invention to treat animals as long as such compounds are not harmful to host animals being treated. Preferred flea peritrophin protein inhibitors of the present invention include, but are not limited to, flea peritrophin protein substrate analogs, and other molecules that bind to a flea peritrophin protein to an allosteric site) in such a manner that the activity of the flea peritrophin protein is inhibited. A flea peritrophin protein substrate analog refers to a compound that interacts with binds to, associates with, modifies) the active site of a flea peritrophin protein. A preferred flea peritrophin protein substrate analog inhibits flea peritrophin protein activity. Flea peritrophin protein substrate analogs can be of any -42o inorganic or organic composition. Flea peritrophin protein substrate analogs can be, but need not be, structurally similar to a flea peritrophin protein natural substrate as long as they can interact with the active site of that flea peritrophin protein. Flea 00 peritrophin protein substrate analogs can be designed using computer-generated structures of flea peritrophin proteins of the present invention or computer structures of flea peritrophin protein's natural substrates. Preferred sites to model include one or more of the active sites of flea peritrophin proteins. Substrate analogs can also be N obtained by generating random samples of molecules, such as oligonucleotides, peptides, peptidomimetic compounds, or other inorganic or organic molecules, and screening such samples for their ability to interfere with interaction between flea peritrophin proteins and their substrates, e.g. by affinity chromatography techniques.

A preferred flea peritrophin protein substrate analog is a flea peritrophin protein mimetic compound, a compound that is structurally and/or functionally similar to a natural substrate of a flea peritrophin protein of the present invention, particularly to the region of the substrate that interacts with the flea peritrophin protein active site, but that inhibits flea peritrophin protein activity upon interacting with the flea peritrophin protein active site.

The present invention also includes a therapeutic composition comprising at least one protective molecule of the present invention in combination with at least one additional compound protective against one or more infectious agents.

In one embodiment, a therapeutic composition of the present invention can be used to protect an animal from flea infestation by administering such composition to a flea in order to prevent infestation. Such administration to the flea and/or animal could be oral, or by application to the animal's body surface topical spot-on, or spraying onto the animal), or by application to the environment spraying).

Examples of such compositions include, but are not limited to, transgenic vectors capable of producing at least one therapeutic composition of the present invention. In another embodiment a flea can ingest therapeutic compositions, or products thereof, present on the surface of or in the blood of a host animal that has been administered a therapeutic composition of the present invention.

O In accordance with the present invention, a host animal an animal that is ,or is capable of being infested with fleas) is treated by administering to the animal a therapeutic composition of the present invention in such a manner that the composition 00 itself a flea peritrophin protein, a flea peritrophin nucleic acid molecule, a flea peritrophin protein inhibitor, a peritrophin protein synthesis suppressor a compound that decreases the production or half-life of a peritrophin protein in fleas), a flea peritrophin protein mimetope, or a anti-flea peritrophin antibody) or a product N1 generated by the animal in response to administration of the composition antibodies produced in response to administration of a flea peritrophin protein or nucleic acid molecule, or conversion of an inactive inhibitor "prodrug" to an active flea peritrophin protein inhibitor) ultimately enters the flea. A host animal is preferably treated in such a way that the compound or product thereof is present on the body surface of the animal or enters the blood stream of the animal. Fleas are then exposed to the composition or product when they feed from the animal. For example, flea peritrophin protein inhibitors administered to an animal are administered in such a way that the inhibitors enter the blood stream of the animal, where they can be taken up by feeding fleas.

The present invention also includes the ability to reduce larval flea infestation in that when fleas feed from a host animal that has been administered a therapeutic composition of the present invention, at least a portion of compounds of the present invention, or products thereof, in the blood taken up by the fleas are excreted by the fleas in feces, which is subsequently ingested by flea larvae. In particular, it is of note that flea larvae obtain most, if not all, of their nutrition from flea feces.

In accordance with the present invention, reducing flea peritrophin protein activity in a flea can lead to a number of outcomes that reduce flea burden on treated animals and their surrounding environments. Such outcomes include, but are not limited to, reducing the viability of fleas that feed from the treated animal, (b) reducing the fecundity of female fleas that feed from the treated animal, reducing the reproductive capacity of male fleas that feed from the treated animal, reducing the viability of eggs laid by female fleas that feed from the treated animal, altering the blood feeding behavior of fleas that feed from the treated animal fleas take up -44less volume per feeding or feed less frequently), reducing the viability of flea N larvae, for example due to the feeding of larvae from feces of fleas that feed from the treated animal, altering the development of flea larvae by decreasing feeding 00 behavior, inhibiting growth, inhibiting slowing or blocking) molting, and/or otherwise inhibiting maturation to adults), and/or altering or decreasing the ability of fleas or flea larvae to digest a blood meal.

Ce¢ In order to protect an animal from flea infestation, a therapeutic composition of Sthe present invention is administered to the animal in an effective manner such that the composition is capable of protecting that animal from flea infestation. Therapeutic compositions of the present invention can be administered to animals prior to infestation in order to prevent infestation as a preventative vaccine) and/or can be administered to animals after infestation. For example, proteins, mimetopes thereof, and antibodies thereof can be used as immunotherapeutic agents.

Therapeutic compositions of the present invention can be formulated in an excipient that the animal to be treated can tolerate. Examples of such excipients include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used. Other useful formulations include suspensions containing viscosity enhancing agents, such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipients can also contain minor amounts of additives, such as substances that enhance isotonicity and chemical stability. Examples of buffers include phosphate buffer, bicarbonate buffer and Tris buffer, while examples of preservatives include thimerosal, or o-cresol, formalin and benzyl alcohol. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration.

In one embodiment of the present invention, a therapeutic composition can include an adjuvant. Adjuvants are agents that are capable of enhancing the immune response of an animal to a specific antigen. Suitable adjuvants include, but are not limited to, cytokines, chemokines, and compounds that induce the production of N cytokines and chemokines granulocyte macrophage colony stimulating factor (GM-CSF), Flt-3 ligand, granulocyte colony stimulating factor (G-CSF), macrophage 0 colony stimulating factor (M-CSF), colony stimulating factor (CSF), erythropoietin (EPO), interleukin 2 interleukin-3 interleukin 4 interleukin 5 (ILinterleukin 6 interleukin 7 interleukin 8 interleukin 10 (IL- 10), interleukin 12 (IL-12), interferon gamma, interferon gamma inducing factor I N (IGIF), transforming growth factor beta, RANTES (regulated upon activation, normal T cell expressed and presumably secreted), macrophage inflamnatory proteins 10 MIP-1 alpha and MIP-1 beta), and Leishmania elongation initiating factor (LEIF)); bacterial components 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 Hunter's TitermaxTM adjuvant (VaxcelTM, Inc. Norcross, GA), Ribi adjuvants (Ribi ImmunoChem Research, Inc., Hamilton, MT); and saponins and their derivatives 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.

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.

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 O administration to an animal, form a solid or a gel in situ. Preferred controlled release formulations are biodegradable bioerodible).

SA preferred controlled release formulation of the present invention is capable 00 of releasing a composition of the present invention into the blood of the treated animal at a constant rate sufficient to attain therapeutic dose levels of the composition. The Stherapeutic composition is preferably released over a period of time ranging from 1 to 12 months. A controlled release formulation of the present invention is capable of effecting a treatment preferably for at least 1 month, preferably for at least 3 months, t preferably for at least 6 months, preferably for at least 9 months, and preferably for at least 12 months.

Acceptable protocols to administer therapeutic compositions in an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art. A suitable single dose is a dose that is capable of treating an animal when administered one or more times over a. suitable time period. For example, a preferred single dose of an inhibitor is from 1 microgram (jig) to 10 milligrams (mg) of the therapeutic composition per kilogram body weight of the animal. Booster vaccinations can be administered from 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 10 jig to 1 mg of the therapeutic composition per kg body weight of the animal is administered from one to two times over a time period of from 2 weeks to 12 months. Modes of administration can include, but are not limited to, subcutaneous, intradermal, intravenous, intranasal, oral, transdermal, intraocular, intranasal, conjunctival, and intramuscular routes. Methods of administration for other therapeutic compounds can be determined by one skilled in the art, and may include administration of a therapeutic composition one or more times, on a daily, weekly, monthly or yearly regimen; routes of administration can be determined by one skilled in the art, and may include any route. A preferred route of administration of an inhibitory compound when administering to fleas is a topical, or "spot-on" formulation administered to the -47- 0 body surface of the animal, so that a flea would encounter the inhibitory compound when attached to the animal; another preferred route of administration of an inhibitory Scompound is an oral formulation that, when fed to an animal, would enter the 0 bloodstream of the animal, which would then be transferred to a flea while feeding from the animal.

A recombinant protein vaccine of the present invention comprises a Srecombinantly-produced flea peritrophin protein of the present invention that is administered to an animal according to a protocol that results in the animal producing S a sufficient immune response to protect itself from a flea infestation. Such protocols can be determined by those skilled in the art.

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 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 not packaged in a viral coat or cellular membrane) nucleic acid as a genetic vaccine as naked DNA or RNA molecules, such as is taught, for example in Wolff et al., 1990, Science 247, 1465-1468) or administering a nucleic acid molecule packaged as a recombinant virus vaccine or as a recombinant cell vaccine the nucleic acid molecule is delivered by a viral or cellular vehicle).

A genetic naked nucleic acid) vaccine of the present invention includes a nucleic acid molecule of the present invention and preferably includes a recombinant molecule of the present invention that preferably is replication, or otherwise amplification, competent. A genetic vaccine of the present invention can comprise one or more nucleic acid molecules of the present invention in the form of, for example, a dicistronic recombinant molecule. Preferred genetic vaccines include at least a portion of a viral genome, a viral vector. Preferred viral vectors include those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, picornaviruses, and retroviruses, with those based on alphaviruses, such as sindbis or Semliki forest virus, species-specific herpesviruses and poxviruses being particularly preferred. Any suitable transcription control sequence can be used, including those disclosed as -48- O suitable for protein production. Particularly preferred transcription control sequences include cytomegalovirus immediate early (preferably in conjunction with Intron-A), Rous sarcoma virus long terminal repeat, and tissue-specific transcription control 00 sequences, as well as transcription control sequences endogenous to viral vectors if viral vectors are used. The incorporation of a "strong" polyadenylation signal is also Spreferred.

t Genetic vaccines of the present invention can be administered in a variety of ways, with intramuscular, subcutaneous, intradermal, transdermal, conjunctival, t intraocular, intranasal and oral routes of administration being preferred. A preferred single dose of a genetic vaccine ranges from 1 nanogram (ng) to 600 Vg, depending on the route of administration and/or method of delivery, as can be determined by those skilled in the art. Suitable delivery methods include, for example, by injection, as drops, aerosolized and/or topically. Genetic vaccines of the present invention can be contained in an aqueous excipient phosphate buffered saline) alone or in a carrier lipid-based vehicles).

A recombinant virus vaccine of the present invention includes a recombinant molecule of the present invention that is packaged in a viral coat and that can be expressed in an animal after administration. Preferably, the recombinant molecule is packaging- or replication-deficient and/or encodes an attenuated virus. A number of recombinant viruses can be used, including, but not limited to, those based on alphaviruses, poxviruses, adenoviruses, herpesviruses, picornaviruses, and retroviruses. Preferred recombinant virus vaccines are those based on alphaviruses (such as Sindbis virus), raccoon poxviruses, species-specific herpesviruses and species-specific poxviruses. An example of methods to produce and use alphavirus recombinant virus vaccines are disclosed in U.S. Patent No. 5,766,602 to Xiong and Grieve.

When administered to an animal, a recombinant virus vaccine of the present invention infects cells within the immunized animal and directs the production of a protective protein or RNA nucleic acid molecule that is capable of protecting the animal from flea infestation as disclosed herein. For example, a recombinant virus vaccine comprising a flea peritrophin nucleic acid molecule of the present invention is O administered according to a protocol that results in the animal producing a sufficient Q^ immune response to protect itself from flea infestation. A preferred single dose of a O recombinant virus vaccine of the present invention is from 1 x 104 to 1 x 108 virus 00 plaque forming units (pfu) per kilogram body weight of the animal. Administration protocols are similar to those described herein for protein-based vaccines, with Ssubcutaneous, intramuscular, intranasal, intraocular, conjunctival, and oral administration routes being preferred.

SA recombinant cell vaccine of the present invention includes recombinant cells O of the present invention that express at least one protein of the present invention.

Preferred recombinant cells for this embodiment include Salmonella, E. coli, Listeria, Mycobacterium, S. frugiperda, yeast, (including Saccharomyces cerevisiae and Pichia pastoris), BHK, CV-1, myoblast G8, COS 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 108 to 1012 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.

The efficacy of a therapeutic composition of the present invention to protect an animal from flea infestation can be tested in a variety of ways including, but not limited to, detection of protective antibodies (using, for example, proteins or mimetopes of the present invention), detection of cellular immunity within the treated animal, or challenge of the treated animal with the fleas to determine whether the treated animal is resistant to infestation. Challenge studies can include direct administration of fleas to the treated animal. In one embodiment, therapeutic compositions can be tested in animal models such as mice. Such techniques are known to those skilled in the art.

As discussed herein, one therapeutic composition of the present invention includes an inhibitor of flea peritrophin protein activity, a compound capable of substantially interfering with the function of a flea peritrophin protein. An inhibitor of flea peritrophin protein activity, or function, can be identified using flea peritrophin O proteins of the present invention. A preferred inhibitor of flea peritrophin protein function is a compound capable of substantially interfering with the function of a flea peritrophin protein and which does not substantially interfere with the function of host 00 animal peritrophin proteins. As used herein, a compound that does not substantially inhibit or interfere with host animal peritrophin proteins is one that, when administered to a host animal, the host animal shows no significant adverse effects attributable to the inhibition of peritrophin and which, when administered to an animal in an effective N manner, is capable of protecting that animal from flea infestation.

One embodiment of the present invention is a method to identify a compound capable of inhibiting flea peritrophin protein activity. Such a method includes the steps of contacting combining, mixing) an isolated flea peritrophin protein of the present invention, with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein has flea peritrophin protein activity, and determining if the putative inhibitory compound inhibits the activity. Such conditions under which a flea peritrophin protein has flea peritrophin protein activity include conditions in which a flea peritrophin protein has a correct three-dimensionally folded structure under physiologic conditions, i.e. physiologic pH, physiologic ionic concentrations, and physiologic temperatures. Putative inhibitory compounds to screen include antibodies (including fragments and mimetopes thereof), putative substrate analogs, and other, preferably small, organic or inorganic molecules.

A preferred method to identify a compound capable of inhibiting flea peritrophin protein activity includes contacting an isolated flea peritrophin protein of the present invention with a putative inhibitory compound under conditions in which, in the absence of the compound, the protein has flea peritrophin protein activity; and determining if the putative inhibitory compound inhibits the activity.

Another embodiment of the present invention is an assay kit to identify an inhibitor of a flea peritrophin protein of the present invention. This kit comprises an isolated flea peritrophin protein of the present invention, and a means for determining inhibition of an activity of flea peritrophin protein, where the means enables detection of inhibition. Detection of inhibition of flea peritrophin protein identifies a putative inhibitor to be an inhibitor of a flea peritrophin protein. Means for determining -51o inhibition of a flea peritrophin protein include, for example, an assay system that 2 detects binding of a putative inhibitor to a flea peritrophin molecule, and an assay system that detects interference by a putative inhibitor of the ability of flea peritrophin 00 protein to hydrolyze a substrate. Means and methods are described herein and are known to those skilled in the art.

The following examples are provided for the purposes of illustration and are not intended to limit the scope of the present invention. The following examples N include a number of recombinant DNA and protein chemistry techniques known to those skilled in the art; see, for example, Sambrook et al., ibid.

Example 1 This Example describes the isolation of RNA from the hindgut and Malpighian tubules (HMT) of Ctenocephalidesfelis and the use of isolated RNA to construct subtracted and unsubtracted cDNA libraries.

Approximately 10,000 hindguts and Malpighian tubules were dissected from equal numbers of cat blood fed and unfed adult C. felis with a male to female ratio of 1 to 4, and total RNA was extracted using a guanidine isothiocyanate lysis buffer and the standard procedure described by Sambrook et al. Poly-A enriched mRNA was purified from total RNA above using a mRNA Purification Kit, available from Pharmacia Biotech, Piscataway, NJ, following the manufacturer's protocol. The same procedures were used to extract total RNA and isolate poly-A enriched mRNA from the dissected C. felis bodies following removal of HMT, referred to hereinafter as "non-HMT mRNA".

Poly-A enriched mRNA was used to construct a cDNA library using subtractive hybridization and suppression PCR as follows. Subtractive hybridization and suppression PCR was conducted using a PCR-SelectTM cDNA Subtraction Kit, available from Clontech Laboratories, Inc., Palo Alto, CA according to the manufacturer's instructions. Briefly, this kit uses subtractive hybridization and suppression.PCR to specifically amplify cDNA sequences that are present in the tester cDNA and absent in the driver cDNA, thus enriching for tester-specific sequences.

The efficiency of the subtraction process can be assessed by semi-quantitative

PCR

and by comparing the ethidium bromide staining patterns of the subtracted and -52unsubtracted samples on agarose gels as described in section V.D. of the manufacturer's protocol. For the semi-quantitative PCR, three genes with mRNAs known to be expressed outside of the HMT tissue were used to test for specific 00 subtraction. These genes encoded putative actin, N-aminopeptidase, and serine protease proteins.

Subtractive hybridization and suppression PCR was conducted under the r^ following conditions. Two micrograms (pg) of HMT mRNA was used as the template for synthesis of the tester material and 2 pg of non-HMT mRNA was used as template for synthesis of the driver material in this reaction. The number of cycles used in the selective amplification steps was optimized using the manufacturer's protocols.

Optimization resulted in the use of 24 rather than the standard 27 cycles of primary PCR in combination with 15 cycles of secondary PCR rather than the standard 12 cycles.

The products from the suppressive PCR reaction were ligated into the pCR@2.1 vector, available from Invitrogen, Carlsbad, CA, using an Original TA Cloning@ Kit, available from Invitrogen. The ligation reaction was then used to transform INVaF' One ShotTM competent cells, available from Invitrogen, which were plated on Luria broth (LB) agar with 50 micrograms per milliliter (pg/ml) ampicillin, available from Sigma-Aldrich Co., St. Louis, MO, and 50 pg/ml 5-bromo-4-chloro-3indoyl P-D-galactopyranoside (X-Gal), available from Fisher Biotech, Fair Lawn, NJ.

Transformed colonies were amplified and the DNA isolated using the standard alkaline lysis procedure described by Sambrook et al., ibid.

Automated cycle sequencing of DNA samples was performed using an ABI PRISMT" Model 377, available from Perkins Elmer, with XL upgrade DNA Sequencer, available from PE Applied Biosystems, Foster City, CA, after reactions were carried out using the PRISMTM Dye Terminator Cycle Sequencing Ready Reaction Kit or the PRISMTM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit or the PRISMTM BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit, available from PE Applied Biosystems, following the manufacturer's protocol, hereinafter "standard sequencing methods". Sequence analysis was performed using SeqLab, using default parameters. Each sequence read was trimmed of vector sequence at either end and -53submitted for a search through the National Center for Biotechnology Information S,(NCBI), National Library of Medicine, National Institute of Health, Baltimore, MD, using the BLAST network. This database includes SwissProt PIR SPupdate 00 GenPept GPUpdate PDB databases. The search was conducted using the xBLAST function, which compares the translated sequences in all 6 reading frames to the protein sequences contained in the database.

An unsubtracted HMT cDNA library was constructed as follows.

Approximately 10,000 HMT tissues were dissected from equal numbers of unfed and cat blood-fed adult C. felis with a male to female ratio of 1:4. Total RNA was extracted using a guanidine isothiocyanate lysis buffer and procedures described in Sambrook et al., followed by isolation using a mRNA purification kit, available from Pharmacia, according to the manufacturer's protocols. The library was constructed with 5 Vg of isolated mRNA using a ZAP-cDNA@ cDNA synthesis kit, and packaged using a ZAP-cDNA@ Gigapack® gold cloning kit, both available from Stratagene, La Jolla, CA. The resultant HMT library was amplified to a titer of 5 x 10 9 plaque forming units per milliliter (pfu/ml). Single clone excisions were performed using the Ex-AssistTM helper phage, available from Stratagene, and used to create double stranded plasmid template for sequencing using the manufacturer's protocols with the following exceptions. Following incubation of the SOLR cells with the cleared phage lysate, the mixture was used to inoculate LB broth, and the mix was incubated overnight and then subjected to mini-prep plasmid preparation and sequencing as described for the subtracted HMT library above.

Example 2 This example describes the production of a C. felis cDNA pool by Rapid Amplification of cDNA Ends (RACE cDNA pool).

Total RNA was extracted from adult fed and unfed fleas as follows.

Approximately 1000 adult fed fleas and 1000 adult unfed fleas were frozen on dry ice and separately ground into powder using a mortar and pestle and total RNA was extracted from each powder as follows. Ten ml of solution D (4 M guanidine isothiocyanate, 25 mM Sodium Citrate pH 7.0, 1.5% Sarcosyl, 0.5 M 2mercaptoethanol) were added to the powder and the suspension was mixed by shaking.

-54- One ml of 2M sodium acetate, pH 4.0 and 3 ml of pH 4.7 phenol/chloroform/isoamyl N alcohol (125:24:1), available from Sigma, were added and the suspension was mixed on a vortex shaker then incubated on ice for 15 minutes. Following incubation, the 00 mixture was centrifuged at 10,000 X g for 20 minutes and the supernatant was removed and extracted twice with pH 4.7 phenol/chloroform/isoamyl alcohol. Next, an equal volume of isopropanol was added to the supernatant and incubated at for 2 hours followed by centrifugation at 10,000 X g for 20 minutes. Following centrifugation, the supernatant was removed and discarded and the pellet was washed in 70% ethanol and allowed to dry at room temperature. The pellet was resuspended in 10 mM Tris 1 mM EDTA pH 8.0. Spectrophotometer analysis indicated that the yield of total RNA from unfed fleas was 1140 pg and the yield from fed fleas was 1500 pg.

Six-hundred pg from each of the fed and unfed adult flea total RNA extractions were combined and mRNA was then extracted using a mRNA Purification Kit, available from Amersham Pharmacia Biotech, Piscataway, NJ, using the manufacture's protocol. Approximately 15-25 pg of mRNA were isolated based on spectrophotometer analysis and ethidium bromide staining. One pg of purified mRNA was used as template to construct a RACE cDNA pool using a SMART T M

RACE

cDNA Amplification Kit, available from Clontech Laboratories, Inc., Palo Alto, CA, according to the manufacture's instructions.

Example 3 This example describes the cloning, sequencing, recombinant protein expression and purification of a C. felis peritrophin-like nucleic acid molecule, referred to herein as PL1. This example also describes the expression of PL1 mRNA in a variety of flea tissues.

A. Isolation of PL1 nucleic acid molecules.

A TA clone from the HMT EST library described in Example 1 was sequenced using standard sequencing methods and shown to have homology to a chitinase-like gene from Bombyx mori (silkworm). This clone was digested with EcoRI to excise an insert 429 nucleotides in length, referred to as peritrophin-like molecule 1 (PL1) nucleic acid molecule nCfPL1 429 The insert was isolated by gel purification using a Gel Purification kit, available from Qiagen, Valencia, CA. Approximately 50 ng of purified nCfPL1 4 29 was used to construct a 32 P a-dATP labeled DNA probe using a Megaprime DNA labeling kit, available from Amersham, using the manufacturer's 00 protocols.

The 32 P ac-dATP labeled probe was used in a plaque lift hybridization procedure Sto isolate a clone from the HMT lambda-ZAP unsubtracted cDNA library described in Example 1 as follows. Filters were hybridized with 1 X 106 counts per minute (cpm) per ml of the probe in 5X SSPE, (see Sambrook et al., ibid.), 1.2% sodium dodecyl sulfate (SDS), 0.1 mg/ml salmon sperm DNA and 5X Denhardt's reagent, (see Sambrook et al., ibid.), at 55°C for 14 hours. The filters were washed as follows: (a) minutes with 5X SSPE and 1% SDS, 10 minutes with 2X SSPE and 1% SDS, 10 minutes with lX SSPE and 0.5% SDS, and 10 minutes with 0.5X SSPE and 1% SDS. All washes were conducted at 55 0 C. Plaques that hybridized strongly to the probe were isolated and subjected to in vivo excision. In vivo excision was performed using the Stratagene Ex-AssistTM helper phage system and protocols, to convert a positive plaque to pBluescriptTM plasmid DNA. Sequencing was conducted using standard sequencing methods following preparation of DNA with a Qiagen QiaprepTM spin mini prep kit using the manufacturer's instructions and restriction enzyme digestion with 1 pl of 20 U/pl each of EcoRI and Xhol, available from New England Biolabs, Beverly, MA. Plaques that hybridized strongly to the probe were isolated and subjected to in vivo excision. In vivo excision was performed using the Stratagene Ex- AssistTM helper phage system and protocols, to convert a positive plaque to pBluescriptTM plasmid DNA, and sequencing was conducted following preparation of DNA with a Qiagen QiaprepTM spin mini prep kit using the manufacturer's instructions and restriction enzyme digestion with 1 pl of 20 U/pl each of EcoRI and XhoI, available from New England Biolabs. A clone was isolated from a primary plaque, containing a nucleic acid molecule of 1096 base pairs, referred to herein as nCfPL1o10 6 the coding strand of which has a nucleotide sequence denoted herein as SEQ ID NO: 1. The complement of SEQ ID NO: 1 is represented herein as SEQ ID NO:3. Sequencing of nCfPL1 42 9 indicated that nCfPL 42 9 shares 100% identity with nucleotides 148 through 576 of SEQ ID NO:1.

0 Translation of SEQ ID NO: 1 suggests that nucleic acid molecule nCfPL1 1 o9 Sencodes a full-length chitin-binding protein of 272 amino acids, referred to herein as PCfCfPL1 272 having an amino acid sequence represented by SEQ ID NO:2, assuming 00 the initiation codon spans from nucleotide 6 through nucleotide 8 of SEQ ID NO:1 and the termination codon spans from nucleotide 822 through nucleotide 824 of SEQ ID NO:1. The coding region encoding PCfPL1 272 is represented by nucleic acid molecule nCfPLIi 1 6 having a coding strand with the nucleic acid sequence represented by SEQ ID NO:4 and a complementary strand with nucleic acid sequence represented by SEQ ID NO:5. The amino acid sequence of PCfPL1 2 72 predicts that PCfPL12 72 has an estimated molecular weight of 30.6 kDa and an estimated pi of 7.3.

Comparison of amino acid sequence SEQ ID NO:2 with amino acid sequences reported in GenBank indicates that SEQ ID NO:2 showed the most homology, i.e., 26% identity, with a Lucilia cuprina peritrophin-44 protein, GenBank Accession No.

407976. Comparison of SEQ ID NO:4 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:4 showed the most homology, 40%, with a Lucilia cuprina peritrophin-44 nucleic acid molecule, GenBank Accession number L25106. Percent identity calculations were performed using the SeqLab software with default parameters.

B. Expression of a PL1 nucleic acid molecule.

A nucleic acid molecule comprising nucleotides 59 through 827 of SEQ ID NO:1, encoding a predicted mature flea PL1, was PCR amplified from the pBluescriptTM clone described above as the template, using sense primer PL1-FE, having nucleotide sequence 5' CGG GAT CCT GCT GAC AGG AAT TCG CCC AC having a BamHI site indicated in bold, designated herein as SEQ ID NO:50, and anti-sense primer PL1-RE, having nucleotide sequence 5' CAT GGT ACC CCT GGT TTA AGC CTT ACT TAG C having a KpnI site indicated in bold, designated herein as SEQ ID NO:51. PCR reactions were performed using standard PCR reaction and thermocycling conditions described herein. The PCR product was digested with BamHI and KpnI and ligated into the vector pTrcHisB, available from Invitrogen, that had been digested with BanHI and KpnI and treated with alkaline phosphatase. The resulting recombinant molecule, referred to herein as pTrc-nCfPL1 76 9 was transformed -57- 0 into E. coli strain BL21, available from Novagen, to form recombinant cell E.

coli:pTrc-nCfPL1 76 9 The recombinant cell was grown under standard conditions and then incubated in the presence of 0.5 pM IPTG to induce expression of recombinant 00 protein, predicted to be a protein of approximately 32 kDa. Expression of protein was confirmed using Coomassie-blue-stained Tris-glycine gel and by Western blot using a T7 tag antibody which showed expression of an 32-kDa protein. The protein product Swas purified by liquid chromatography using a HiTrapTM chelating column charged N with NiCl,, available from Pharmacia, and was shown to contain the His tag of the r vector when subjected to automated protein sequencing by Edman degradation.

C. Northern Blot Analysis A Northern Blot analysis was conducted as follows to determine whether PL1 is expressed exclusively in HMT tissues. HMT tissues were dissected from 1000 adult cat blood-fed C. felis having a male to female ratio of 1:4. Total RNA was separately extracted from HMT tissues and the HMT-less carcasses that resulted from these dissections as follows. The tissues were frozen at -80°C, ground into a powder with a mortar and pestle, and the powders were equally divided into four 2-ml eppendorf tubes each containing 1 ml of lysis buffer. The lysis buffer contained 4 M guanidinium thiocyanate, 25 mM sodium citrate, pH 7.0, 3% sarcosyl, 0.5M 2mercaptoethanol, 0.1% antifoam, and 1 mM aurintricarboxylic acid, all available from Sigma Chemical Corporation, St. Louis, MO. After mixing, the tubes were spun at 14,000 rpm for 2 minutes and the supernatants were transferred to separate 2 ml eppendorf tubes containing 250 gl of phenol, available from Aldrich, Milwaukee,

WI.

After mixing, the tubes were spun at 14,000 rpm for 5 minutes and the supernatants were transferred to new 2-ml tubes. This process was repeated 3 times until no proteinaceous matter was visible at the phenol/lysis buffer interface, then 250 gl of chloroform was added to each tube and the contents mixed and spun at 14,000 rpm for minutes followed by transferring the supernatant to a new tube. A volume of isopropanol equal to the volume of the supernatant was added to each tube and the tubes placed on ice for 5 minutes. The tubes were then spun at 14,000 rpm at room temperature for 15 minutes, the supernatants were removed and discarded and the remaining RNA pellets were washed with 70% ethanol and dried. The RNA pellets O were resuspended in 100 pl of TE (10 mM Tris, 1 mM ethylenediaminetetraacetic acid (EDTA)). The quantity of RNA in each tube was then determined using a spectrophotometer.

0o Approximately 10 pg of each RNA was added to separate tubes containing 18.75 pI of loading buffer, which consists of 50% formamide, 16% formaldehyde, 17% water, 7% glycerol, 1 X MOPS buffer (a 1:20 dilution of 0.4 M 93-[N- _r morpholino]propanesulfonic acid (MOPS), 0.1 M sodium acetate, and 20 mM EDTA), pl ethidium bromide, and 10 gl bromophenol blue dye, all available from Sigma.

S The tubes were heated to 95°C for 2 minutes then placed on ice. The RNA samples were separated by gel electrophoresis on a 1.5% agarose gel with 3.2% formaldehyde and 1 X MOPS buffer; the gel was then soaked in water for 30 minutes prior to transfer to remove excess formaldehyde. The gel was then transferred using standard techniques, described by Sambrook et al., ibid, with 10 X SSPE as the transfer buffer onto Nytran@ nylon membrane, available from Schleicher and Schuell Inc., Keene, NH. The membrane was UV cross-linked using the Stratalinker®, available from Stratagene, then prehybridized at 42 0 C in 50% formamide, 5X SSPE, 1.2% SDS, Denhardt's reagent, 2.5 mM EDTA, and 100 pg/ml salmon sperm DNA. A probe comprising the PL1 nucleic acid molecule, nCfPL1 1 i 9 6 was labeled with a- 3

P-ATP

using a DNA labeling kit, available from Amersham and added to the buffer at a concentration of approximately 1 x 106 cpm/ml, and allowed to hybridize for 18 hours at 42°C. The blot was then washed as follows: 10 minutes at 42°C in 4X SSPE and 1% SDS; 10 minutes at 42 0 C in 2X SSPE and 1% SDS; 10 minutes at 42 0 C with SSPE and 0.5X SDS; and 10 minutes at 42 0 C with 0.25X SSPE and 0.25% SDS. The blot was then exposed to film for 1 hour, and the film was developed using standard procedures. Analysis of the developed film revealed that PL1 mRNA was present in HMT tissues but was not present in non-HMT tissues.

Northern Blot analysis was also conducted to determine whether PL1 mRNA is expressed only in certain stages of the flea life cycle and whether PL1 mRNA expression is influenced by feeding. Total RNA was extracted as described above from 1000 fleas at each of the following flea life stages; eggs; first instar larvae; third instar larvae; wandering larvae and pupae as well as from 1000 adult fleas under the O following feeding conditions: unfed; fed on cat blood for 15 minutes; fed on cat blood for 2 hours; fed on cat blood for 8 hours; and fed on cat blood for 24 hours. Each RNA sample was separated by gel electrophoresis, transferred to nylon membrane and 0 hybridized with a- 3 2 P-ATP labeled nCfPL1 4 2 probe as described above. Analysis of the developed film revealed that PL1 mRNA was detected in all adult fleas tested regardless of feeding conditions but was not detected in any of the non-adult life stages.

SExample 4 This Example describes the further isolation and characterization of a Peritrophin-like cDNA nucleic acid molecules, referred to herein as PL2.

A cDNA designated clone 2232-23 was isolated from the unsubtracted HMT library as described in Example 1, denoted herein as SEQ ID NO:6. Analysis of clone 2232-23 indicated that the cDNA, denoted nCfPL2 5 is 445 nucleotides in length.

Translation of the coding strand of nCfPL244 suggests that nucleic acid molecule nCfPL244 5 encodes a partial-length Peritrophin-like protein of 113 amino acids, referred to herein as PCfPL2,, 3 assuming a stop coding spanning nucleotides 342 through 344 of nCfPL24.

Additional coding sequence corresponding to the 5' end of nCfPL2s was isolated by PCR performed using a RACE cDNA pool prepared as described in Example 2 as template. A first PCR reaction was performed using reverse primer PL2-R1, which is complementary to nucleotides 167 through 187 of the nCfPL2 44 cDNA, having a nucleic acid sequence 5' GTC TGG AAG CTC AGG AAG AGG 3', denoted herein as SEQ ID NO:52, in conjunction with forward Adapter Primer 1, having a nucleic acid sequence 5' CCA TCC TAA TAC GAC TCA CTA TAG GGC denoted herein as SEQ ID NO:53, under the following thermocycling conditions: 94 0 C for 30 seconds, 5 cycles of 94°C for 10 seconds and 72 0 C for 4 minutes, 5 cycles of 94 0 C for 10 seconds and 70°C for 4 minutes, and 25 cycles of 94°C for 10 seconds then 68'C for 4 minutes The product of this reaction was diluted 1:50 and used as template for a second PCR reaction as follows. Forward adapter primer 2, having nucleic acid sequence 5' ACT CAC TAT AGG GCT CGA GCG GC 3', denoted herein as SEQ ID NO:54, was used with reverse primer PL2-R2, which is (-oU- O complementary to nucleotides 29-52 of the nCfPL24 cDNA, having a nucleic acid sequence 5' GTA ATA TGC GTG ACA ATC GTG TGG denoted herein as SEQ ID using the thermocycling conditions described for the first PCR reaction. The 0 resulting product was gel purified to reveal a distinct band corresponding to a nucleic acid molecule of approximately 900 bp in length. The fragment was then ligated into e the pCR II TA Cloning vector, available from Qiagen, and sequenced using an ABI PRISM 377 automatic DNA Sequencer. Sequencing revealed that nucleotides 791- 835 of the fragment had 100% identity with nucleotides 1-45 of the nCfPL2,4 cDNA.

SThe 900 nucleotide and 445 nucleotide sequences were aligned to form a contiguous 10 sequence, denoted nCfPL2 1 27 9 which is 1279 nucleotides in length, having a coding strand with nucleic acid sequence SEQ ID NO:7 and a complementary sequence having SEQ ID NO:8. Translation of SEQ ID NO:7 suggests that nucleic acid molecule nCfPL2, 2 7 encodes a non full-length Peritrophin-like protein of 391 amino acids.

In order to isolate additional sequence 5' to SEQ ID NO:7, nested PCR reactions were performed using the RACE cDNA pool as template. For the first PCR, forward adapter primer API (SEQ ID NO:53) was used with reverse primer PL2-R1 (SEQ ID NO:52) under standard PCR reaction conditions and the following thermocycling conditions: 94C for 1 minute, 5 cycles of 94°C for 20 seconds and 70'C for 1 minute, 5 cycles of 94'C for 20 seconds and 68'C for 1 minute, (4) cycles of 94'C for 20 seconds and 66*C for 1 minute. The products of this reaction were diluted 1:50 in water and used as template for the second, nested PCR. The second PCR reaction used forward adapter primer AP2 in conjunction with reverse primer PL2-R5, which is complementary to nucleotides 70-93 of SEQ ID NO:7, having a nucleotide sequence 5' CGG TGC AAG TTA TAG AAC CTT CCG 3', denoted herein as SEQ ID NO:56 under standard PCR reaction conditions using the following thermocycling conditions: 94'C for 1 minute, 5 cycles of 94°C for seconds and 70*C for 1 minute, 5 cycles of 94*C for 20 seconds and 68'C for 1 minute, 40 cycles of 94C for 20 seconds and 66'C for 1 minute. The products of this reaction were separated by agarose gel electrophoresis and a band approximately 279 nucleotides in length was excised from the gel and purified. The fragment, -61- O referred to as nCfPL'7,, having a coding nucleic acid sequence designated SEQ ID NO:9 and a complementary sequence designated SEQ ID NO: 10, was then ligated into Sthe pCROII TA Cloning vector, available from Qiagen, and sequenced as described Sabove. Sequencing revealed that nucleotides 228-279 of nCfPL2 27 9 were identical to nucleotides 42-93 of SEQ ID NO:7, however, nucleotides 186-228 of nCfPL2 2 79 had Sno significant similarity to SEQ ID NO:7. This discrepancy may be the result of alternative RNA splicing or may be an artifact of the cDNA pool. To determine the reason for this discrepancy, additional fragments corresponding to this region were isolated by PCR from flea cDNA libraries from adult midguts, hindgut and Malpighian 10 tubules and mixed instar larvae using techniques described herein. Sequence analysis of fragments obtained from these libraries revealed that these fragments were identical in sequence to the sequence of nCfPL2 2 7 therefore, the region of SEQ ID NO:7 which did not align to nCfPL2 2 79 was deemed to be an artifact and was not used in subsequent alignments.

The PL2 sequences described above were aligned to form a contiguous sequence, denoted nCfPL2 1 4 65 which is 1465 nucleotides in length, having a coding strand with nucleic acid sequence SEQ ID NO: 11 and a complementary sequence having SEQ ID NO:13. Translation of SEQ ID NO:11 suggests that nucleic acid molecule nCfPL2 1 46 encodes a full-length Peritrophin-like protein of 453 amino acids, referred to herein as PCfPL2 4 5 3 having an amino acid sequence represented by SEQ ID NO:12, assuming an initiation codon spanning from nucleotide 3 through nucleotide of SEQ ID NO: 11 and a termination codon spanning from nucleotide 1362 through nucleotide 1364 of SEQ ID NO: 11. The coding region encoding PCfPL24, 3 is represented by nucleic acid molecule nCfPL2 3 5 g, having a coding strand with the nucleic acid sequence represented by SEQ ID NO: 14 and a complementary strand with nucleic acid sequence represented by SEQ ID NO: 15. The amino acid sequence of SEQ ID NO: 12, predicts that PCfPL2 4 3 has an estimated molecular weight of 49 kDa and an estimated isoelectric point (pI) of 4.7.

Comparison of amino acid sequence SEQ ID NO: 12 with amino acid sequences reported in GenBank indicates that SEQ ID NO: 12 showed the most homology, 28% identity, with a Drosophila melanogaster locus AE003474 protein -62- 0 (Accession AAF47629). Comparison of SEQ ID NO: 14 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:14 showed the most homology, i.e., identity, with a Penaeus semisulcatus (a crustacean) peritrophin-like protein 1 00 cDNA (Accession AF095580). Percent identity calculations were performed using the SeqLab software with default parameters.

Example This Example describes the further characterization and expression of a flea Peritrophin-like sequence cDNA, referred to herein as PL3.

A. Isolation of PL3 nucleic acid molecules.

10 A cDNA designated clone 2240-17 was isolated from the unsubtracted HMT library as described in Example 1. Analysis of clone 2240-17 indicated that the cDNA, denoted nCfPL3 3 is 387 nucleotides in length, having a coding strand with nucleic acid sequence SEQ ID NO:16 and a complementary sequence having SEQ ID NO:18. Translation of SEQ ID NO:16 suggests that nucleic acid molecule nCfPL3 3 encodes a full-length Peritrophin-like protein of 81 amino acids, referred to herein as PCfPL3 8 having an amino acid sequence represented by SEQ ID NO:17, assuming the initiation codon spans from nucleotide 20 through nucleotide 22 of SEQ ID NO:16 and the termination codon spans from nucleotide 263 through nucleotide 265 of SEQ ID NO: 16. The coding region encoding PCfPL3 8 1 is represented by nucleic acid molecule nCfPL3 2 43 having a coding strand with the nucleic acid sequence represented by SEQ ID NO:19 and a complementary strand with nucleic acid sequence represented by SEQ ID NO:20. The amino acid sequence of SEQ ID NO:17, predicts that PCfPL3si has an estimated molecular weight of 9.1 kDa and an estimated isoelectric point (pI) of 3.64.

Comparison of amino acid sequence SEQ ID NO:17 with amino acid sequences reported in GenBank indicates that SEQ ID NO: 17 showed the most homology, 34.2% identity, with a Anopheles gambiae peritrophin 1 protein (Accession AAC39127). Comparison of SEQ ID NO: 19 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:19 showed the most homology, i.e., 37% identity, with a Anopheles gambiae chloride intracellular channel 2 (Accession -63o AF030431). Percent identity calculations were performed using the SeqLab software with default parameters.

B. Expression of a PL3 protein.

SIn order to express a PL3 protein, the entire coding region was amplified by PCR and then ligated into the E. coli expression vector pTrcHisB, available from Invitrogen, as follows. Forward primer PL3FE, which corresponds to nucleotides r 93 of SEQ ID NO: 16, having the sequence 5' CGG GAT CCC GAA TAT GCT GAC GTA GAT GTG TG denoted SEQ ID NO:57, and having aBamHI restriction r endonuclease site indicated in bold, was used in conjunction with reverse primer PL3RE, which is complementary to nucleotides 245-269 of SEQ ID NO:16, having the sequence 5' GGA ATT CTG TTT TAT TCT GGT TGG TAA CAT TC denoted herein as SEQ ID NO:58 and having an EcoRI restriction endonuclease site indicated in bold, in a PCR reaction using SEQ ID NO: 16 as the template under standard PCR reaction conditions and the following thermocycling conditions: 94 0 C for seconds, 25 cycles of 94°C for 10 seconds, 55 0 C for 10 seconds and 72 0 C for 3 minutes. The reaction product.was separated on a 1.5% agarose gel, and a band corresponding to an approximately 200 nucleotide molecule, as visualized by agarose gel electrophoresis and ethidium bromide staining, was cut from the gel and purified using a QIAquick Gel Extraction Kit, available from Qiagen.

The product of the PCR reaction was the digested with BamHIl and EcoRI restriction endonucleases, available from New England BioLabs, Inc. for 18 hours at 37'C, purified using the QIAquick Nucleotide Removal Kit, available from Qiagen, and ligated into the vector pTrcHisB which had been similarly digested, treated with shrimp alkaline phosphatase, available from New England BioLabs, Inc., for minutes at 37 0 C, and purified. Following standard transformation procedures into E.

coli BL-21 competent cells, a bacterial clone containing the plasmid pTrcPL3 2 ,0 was isolated. DNA sequence analysis of the clone confirmed that nucleotides 70 through 269 of SEQID NO: 16 had been successfully ligated into the pTrcHisB expression vector in frame with the N-terminal T7 Tag epitope encoded by the vector. The recombinant protein encoded thereby is predicted to be 97 amino acids in length -64- S(including the T7 Tag) and have a molecular mass of 10.9 kDa, including the T7 Tag, and have a pI of 4.08.

SThe 97 amino acid recombinant PL3 protein described above was expressed as 00 follows. Five mis of Luria broth were innoculated with a glycerol stock of E. coli BL- 21 competent cells, available from Novagen, Madison, WI, that had been transformed with the pTrcPL3 2 0 plasmid prepared as described above and allowed to grow overnight at 37 0 C under selection with 100 pg/ml ampicillin. A 1-ml aliquot of this culture was then used to inoculate 10 mis of fresh Luria broth containing 100 pg/ml ampicillin and the culture was allowed to grow to an approximate OD reading of A 1 ml aliquot of the culture was removed, the cells were pelleted by centrifugation and the supernatant discarded. The cells were resuspended in a solution of 100 ul PBS and 100 pl of 2X SDS-PAGE loading buffer (100 mM Tris pH 6.8, 4% SDS, glycerol, 0.02% bromophenol blue, and 10% 2 -mercaptoethanol). Following removal of the 1 ml aliquot described above, IPTG was added to the remaining 9 ml culture to a final concentration of 5 mM of IPTG, the culture was incubated at 37°C for an additional 60 minutes, 1 ml was removed and the OD measured at approximately 0.6.

The cells in this 1 ml sample were then pelleted by centrifugation and resuspended in a solution of 120 pl of PBS and 120 pl of SDS-PAGE loading buffer. Equal volumes of the IPTG-induced and uninduced lysates were loaded onto a 14% Tris-Glycine

SDS-

PAGE gel, available from Novex, San Diego, CA. Following electrophoresis, the proteins were transferred from the SDS-PAGE gel to a nitrocellulose membrane and a Western blot analysis was performed using the T7 tag antibody, available from Novagen, which revealed an approximately 18 kDa protein was induced by IPTG. The fact that the recombinant nCfPL3 200 protein ran at a higher molecular weight than predicted is consistent with previous published results for other peritrophin proteins, and is thought to be due in part to the characteristically low pi of these proteins; See Tellam et al., 1999, Insect Biochemistry and Molecular Biology, 29:87-101. Sequence analysis of this protein indicates that it contained the N-terminal T7 Tag encoded by the vector.

Four flasks, each containing 1 liter of Luria broth with 100 pg/ml ampicillin were inoculated with a starter culture of 5 ml of E. coli BL-21 cells transformed with the pTrcnCfPL3 2 00 plasmid as described above. The cultures were allowed to grow at S37 0 C until the optical density reached approximately 0.500, at which time a 1 ml aliquot was removed from each flask as the pre-induction sample. IPTG was added to 0 each 1 liter flask to a final concentration of 0.5 mM and the cultures allowed to grow at 37°C for 135 additional minutes, at which time a 1 ml aliquot was removed from each flask as the post-induction sample. The 1 ml aliquots were centrifuged, the supernatants were discarded and the pellets were resuspended in 100I l 2X SDS-PAGE Sloading buffer per each 0.5 optical density units measured. The pre-induction and post induction samples were then tested for recombinant PL3 protein expression using standard Western blot techniques and the T7 Tag antibody. A protein running at approximately 18 kDa was detected in the post-induced but not in the pre-induced samples.

The cells from the remaining 4 liters of culture were centrifuged, the supernatants were discarded and the cell pellets were combined and resuspended in 120 mis of buffer A (50 mM Tris, PH 8.0, 20 mM NaC1, 1 mM phenylmethylsulfonyl fluoride (PMSF)). The sample was then passed through a microfluidizer five times then rocked at 4 0 C for 20 minutes. The sample was then centrifuged for 30 minutes and the supernatant collected. Western blot analysis of the supernatant showed that the recombinant nCfPL3 2 0 protein was soluble in the first buffer A extraction. The buffer A supernatant containing the recombinant nCfPL320 protein was then further purified by a nickel column, a Q2 anion exchange chromatography column, and cation exchange chromatography, using techniques well known to those of skill in the art.

Example 6 This Example describes the characterization, expression, and Northern Blot analysis of a Peritrophin-like sequence cDNA, referred to herein as PL4.

A. Isolation of PL4 nucleic acid molecules A cDNA designated clone 2244-71 was isolated from the unsubtracted HMT library as described in Example 1. Analysis of clone 2244-71 indicated that the cDNA, denoted nCfPL4 9 6 is 960 nucleotides in length, having a coding strand with nucleic acid sequence SEQ ID NO:21 and a complementary sequence having SEQ ID NO:22. Translation of SEQ ID NO:21 suggests that nucleic acid molecule nCfPL4 9 6 encodes a partial-length Peritrophin-like protein of 285 amino acids. Additional Ssequence 5' to nCfPLI,6 was isolated by PCR using the RACE cDNA pool described in Example 2 as the template, as follows. Adapter Primer 1, i.e. SEQ ID NO:53, was 0used as the forward primer in conjunction with reverse primer PL4-R1, which is complementary to nucleotides 229-251 of SEQ ID NO:21, having a nucleic acid sequence 5' GAT ATC CAC TTT GAT CAG CGC AC denoted herein as SEQ ID NO:59 in a PCR reaction under standard PCR reaction conditions and the following (N thermocycling conditions: 94°C for 30 seconds, 5 cycles of 94 0 C for seconds and 72 0 C for 4 minutes, 5 cycles of 94 0 C for 10 seconds and 70 0 C for 4 minutes, 25 cycles of 94°C for 10 seconds then 68 0 C for 4 minutes. The products of this reaction were diluted 1:50 and used as template in a second PCR reaction using Adapter Primer 2, i.e. SEQ ID NO:54, as the forward primer and reverse primer PL4- R2, which is complementary to nucleotides 58-78 of SEQ ID NO:21, having a nucleic acid sequence 5' GGT ACT ACT CCT GGT GCG GGC denoted herein as SEQ ID NO:60, using the thermocycling conditions described for the first PCR reaction. The products of this reaction were gel purified as previously described and the fragment was ligated into the pCR II TA Cloning vector, available from Qiagen, and sequenced to reveal a fragment of approximately 150 nucleotides in length. Sequence analysis revealed that nucleotides 68-146 of the fragment had 100% identity with nucleotides 1-79 of nCfPAL49o. The two sequences were aligned to form a contiguous sequence of 1029 nucleotides in length, referred to as nCfPL4t 29 having a coding strand with SEQ ID NO:23 and a complementary strand having SEQ ID NO:24. However, the contiguous sequence did not appear to encode a starting methionine in the predicted protein sequence; thus, a second attempt to isolate the remaining coding sequences at the 5' end was performed as follows. A first PCR reaction was performed with Adapter Primer 1 (SEQ ID NO:53) as the forward primer and PL4-R2 (SEQ ID as the reverse primer using the RACE cDNA pool as the template under the thermocycling conditions described above. The products of this reaction were diluted 1:50 and used as the template in a second PCR reaction which used Adapter Primer 2 (SEQ ID NO:54) as the forward primer and reverse primer PL4-R4, which is complementary to nucleotides 58-80 of SEQ ID NO:23, having the nucleic acid -67sequence 5' CCG TCG ACA TTA AAC TCA CCA TC denoted SEQ ID NO:61, C under the thermocycling conditions described for the first PCR reaction. The products of this reaction were gel purified as previously described and the fragment was ligated 0into the pCR II TA Cloning vector, available from Qiagen, and sequenced to reveal a fragment of approximately 100 nucleotides in length. Sequence analysis revealed that nucleotides 21-101 of the fragment had 100% identity with nucleotides 1-81 of SEQ ID NO:21. The two sequences were aligned to form a contiguous sequence that is N 1048 nucleotides in length, referred to herein as nCfPL4, 4, having a coding strand with SEQ ID NO:25 and a complementary strand with SEQ ID NO:27. Translation of SEQ ID NO:25 suggests that nucleic acid molecule nCfPL4A, 4encodes a full-length Peritrophin-like protein of 285 amino acids, referred to herein as PCfPL4 285 having an amino acid sequence represented by SEQ ID NO:26, assuming the initiation codon spans from nucleotide 19 through nucleotide 21 of SEQ ID NO:25 and the termination codon spans from nucleotide 874 through nucleotide 876 of SEQ ID NO:25. The coding region encoding PCfPL4 2 SS is represented by nucleic acid molecule nCfPL4Ac, having a coding strand with the nucleic acid sequence represented by SEQ ID NO:28 and a complementary strand with nucleic acid sequence represented by SEQ ID NO:29. The amino acid sequence of SEQ ID NO:26 predicts that PCfPL4 2 has an estimated molecular weight of 31.4 kDa and an estimated isoelectric point (pI) of 6.99.

Comparison of amino acid sequence SEQ ID NO:26 with amino acid sequences reported in GenBank indicates that SEQ ID NO:26 showed the most homology, 31.5% identity, with a Drosophila melanogaster Gasp precourser (Accession AAD09748). Comparison of SEQ ID NO:28 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:28 showed the most homology, 39.4% identity, with a Drosophila melanogaster Gasp precourser (Accession #AF070734). Percent identity calculations were performed using the SeqLab software with default parameters.

B. Northern Blot Analysis A Northern Blot analysis was conducted as described in Example 3 to determine whether PL4 mRNA is expressed only in certain life stages of the flea life cycle and whether PL4 mRNA is expressed only in HMT tissue. Total RNA was extracted from eggs, first instar larvae, third instar larvae, and wandering larvae, C pupae, unfed adults, and adults fed on cat blood for 0.25, 2, 8, or 24 hours, respectively. In addition, total RNA was extracted from hindguts and Malpighian o tubules extracted from 24-hour cat blood-fed adult fleas, and from the remaining body parts following the removal of hindguts and Malpighian tubules. Each RNA sample was separated by gel electrophoresis, transferred to nylon membranes and hybridized with a- 3 2 P-ATP labeled nCfPLA96 0 using the conditions described in Example 3.

The results of the Northern blot assay were complex. Although stringent conditions were used, several bands with distinct expression patterns were seen. An approximately 1600 nucleotide message was detected in the egg, first instar, third instar and wandering larval stages only. An approximately 1500 nucleotide message was detected in all lifestages and adult fed timepoints, but with the strongest signals in the egg, first instar larval, and unfed adult stages. A third message, of approximately 1200 nucleotides, was detected in the egg, first instar larval, pupal, and adult lifestages, including all unfed and fed adult timepoints. All three of the messages detected were seen only in the HMT tissues, and were not detected in the carcass tissues.

The detection of three mRNAs instead of one may be the result of the expression of three highly homologous transcripts. It has been reported in the literature that peritrophin gene families have been found that consist of a number of highly related genes, See Schorderet et al., 1998, Insect Biochemistry and Molecular Biology 28, 99-111. It is possible that these transcripts represent the products of such a family or that the messages are the RNA products of alternative splicing from a single gene locus.

The coding region of SEQ ID NO:25, was PCR amplified from the RACE cDNA pool described above as the template, using sense primer PL4FE, having nucleotide sequence 5' CGG GAT CCT TAT GAT GGT GAG TTT AAT GTC G 3', which corresponds to nucleotides 75-96 of SEQ ID NO:25, having a BamHI site indicated in bold, designated herein as SEQ ID NO:62, and anti-sense primer PL4RE, having nucleotide sequence 5' GGG GTA CCT TAA TAT AAT TTA GGT TTC CTC TCG C which is complementary to nucleotides 851-876 of SEQ ID NO:25, having -69- 0 a KpnI site indicated in bold, designated herein as SEQ ID NO:63. PCR reactions were performed using the following amplification cycles: one cycle at 94°C for thirty seconds; thirty cycles at 94 0 C for twenty seconds, 68 C for thirty seconds, 00 and 72°C for three minutes; and one cycle at 72°C for five minutes, in reactions containing 2.5 mM MgC12, 0.2 mM dNTPs, 1 gM of each primer, 0.5 pI of 5U/pl Taq polymerase, 1 pl of 1 pg/pl template, and 3 pl of 10X Taq buffer. The products of this reaction were separated on a 1.5% agarose gel, and the appropriate band cut from the Sgel and purified using the QIAquick Gel Extraction Kit, available from Qiagen, Valencia, CA. The resulting nucleic acid molecule, referred to herein as nCfPL4 8 0 2, is 0 10 approximately 802 nucleotides in length, having a coding strand designated SEQ ID and a complementary strand designated SEQ ID NO:32. Translation of SEQ ID NO:30 indicates that SEQ ID NO:30 encodes an approximately 266 amino acid protein, designated SEQ ID NO:31, assuming a first codon at nucleotides 2 through 4 and a stop codon at nucleotides 800 through 802 of SEQ ID The purified PCR product was digested with BamHI and KpnI, purified using a QIAquick Nucleotide Removal Kit, available from Qiagen, and ligated into the vector pTrcHisB, available from Invitrogen, that had been similarly purified, digested with BamnHI and KpnI and treated with alkaline phosphatase. DNA sequence analysis of the clone confirmed that nucleotides 75 through 876 of SEQ ID NO:25 had been successfully ligated into the pTrcHisB expression vector in frame with the N-terminal T7 Tag epitope encoded by the vector. The resulting recombinant molecule, referred to herein as pTrc-nCfPL4,A (which includes the T7 tag), was transformed into E. coli strain BL21, available from Novagen Inc., Madison, WI, to form recombinant cell E.

coli:pTrc-nCfPL4 8 9 4 Recombinant molecule pTrc-nCfPL4 8 94 is predicted to encode a protein including the T7 Tag of 298 amino acids in length, having a molecular mass of 32.8 kDa, and a pi of 5.97.

Recombinant cell E. coli:pTrc-nCfPL4, 9 4 was grown as described in Example and then incubated in the presence of 0.5 mM isopropylthio-p-galactoside (IPTG) to induce expression of recombinant protein PCfPL4 2 98 Expression was confirmed by Western blot using a T7 tag antibody, available from Novagen, which showed expression of an 48-kDa protein. Sequence analysis of PCfPL4 2 indicates that it contained the N-terminal T7 Tag encoded by the vector. The fact that PCfPL4 2 A ran at a higher molecular weight than predicted is consistent with previous published results for other peritrophin proteins, and is thought to be due in part to the characteristically 0 low pi of these proteins, See Tellam et al. ibid.

Recombinant protein PCfPL4 2 9, was produced and purified as follows. Four flasks containing 1 liter each of Luria broth with 100 ug/ml ampicillin were innoculated with E. coli BL21 cells transformed with the pTrc PCfPL4 2 8 plasmid as Sdescribed above. The cultures were allowed to grow at 370 until the optical density (OD6) reached approximately 0.500 then a one ml aliquot was removed as the preinduction sample. Next, IPTG was added to a final concentration of 0.5 mM and the cultures allowed to grow at 37 0 C for 135 minutes. A one-ml aliquot was then removed as the post-induction sample. Both one-ml aliquots were centrifuged to pellet the cells. The cells were resuspended in 100 pl 2X SDS-PAGE loading buffer for each optical density unit measured. The pre-induction and post induction samples were then tested for expression of PCfPL4 2 8 using by Western blot with the T7 Tag antibody described previously. A protein running at approximately 48 kDa was detected in the post-induced but not in the pre-induced samples.

The cells from the remaining 4 liters of culture were pelleted by centrifugation and resuspended in 120 ml of Buffer A. The sample was passed through a microfluidizer five times, rocked at 4* for 20 minutes, then centrifuged for 30 minutes and the supernatant collected. Western blot analysis of the supernatant showed that the recombinant protein PCfPL42 9 was soluble in Buffer A. Protein contained in the Buffer A supernatant was then purified by nickel ion exchange chromatography and hydroxyapatite chromatography.

Example 7 This Example describes the characterization and expression of a flea Peritrophin-like sequence cDNA, referred to herein as cDNA nucleic acid molecules 2109-28, 2234-75, 2241-70, 2164-22, 2183-05, and 2162-56, designated SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, respectively, were isolated from the unsubtracted HMT library as described in Example 1. Sequence analysis revealed that -71these clones contain overlapping sequence to form a 1513-nucleotide cDNA consensus sequence denoted nCfPL55, 3 having a coding strand designated SEQ ID NO:39 and a complementary strand designated SEQ ID NO:41. Translation of the coding strand of 00 nCfPL5t 5 13 suggests that nucleic acid molecule nCfPL5, n 1 encodes a partial-length Peritrophin-like protein of 339 amino acids, referred to herein as PCfPL5 3 39 with an amino acid sequence designated SEQ ID NO:40, assuming a stop codon at nucleotides 1018-1020 of nCfPL5 1 5 13 Protein PCfPL5 339 has a predicted weight of 36.5 kDa and a N predicted pi of 6.98.

Additional coding sequence 5' tonCfPL5 1 s 1 3 was isolated by PCR performed using a RACE cDNA pool prepared as described in Example 2 as template. A first PCR reaction was performed using reverse primer PL5-RI, which is complementary to nucleotides 513-533 of the nCfPL 1 5 3 cDNA, having nucleic acid sequence 5' GCG CAT GTA AAA CGA CCC ACG denoted herein as SEQ ID NO:64, in conjunction with the universal primer mix from the SMART RACE cDNA amplification kit described in Example 2, which contains two primers, the first having nucleic acid sequence 5' CTA ATA CGA CTC ACT ATA GGG CAA GCA GTG GTA ACA ACG CAG AGT and the second having nucleotide sequence 5' CTA ATA CGA CTC ACT ATA GGG C denoted herein as SEQ ID NO:65 and SEQ ID NO:66 respectively, under the following thermocycling conditions: 94 0 C for 30 seconds, 35 cycles of 94 0 C for 30 seconds, 68C for 30 seconds and 72 0 C for 3 minutes, (3) 72 0 C for 7 minutes. The resulting product was gel purified to reveal a distinct band corresponding to nucleic acid molecule of approximately 850 nucleotides in length, referred to herein as nCfPL5 8 5o Fragment nCfPL5 8 5 0 was purified using a QIAquick Gel Extraction Kit, ligated into the pCR II TA Cloning vector, available from Invitrogen, and sequenced using an ABI PRISM 377 automatic DNA Sequencer, available from Perkin Elmer. Sequencing revealed that nucleotides 320-852 of 8 5 had 100% identity with nucleotides 1-533 of the nCfPL5, 1 3 cDNA. The two sequences were aligned to form a contiguous sequence, denoted nCfPL5, 832 which is 1832 nucleotides in length, having a coding strand with nucleic acid sequence SEQ ID NO:42 and a complementary sequence having SEQ ID NO:44.

Translation of SEQ ID NO:42 suggests that nucleic acid molecule nCfPL5 C0

L

18 3 2 encodes a full-length Peritrophin-like protein of 397 amino acids, referred to herein as 397 having an amino acid sequence represented by SEQ ID NO:43, assuming 0 a start codon spanning nucleotides 146 through 148 and a stop codon spanning nucleotides 1337 through 1339 of SEQ ID NO:42. The coding region encoding 39 7 is represented by nucleic acid molecule nCfPL5 1 1 9 having a coding strand with the nucleic acid sequence represented by SEQ ID NO:45 and a N complementary strand with nucleic acid sequence represented by SEQ ID NO:46. The amino acid sequence of PCfCfPL5 397 predicts that PCfCfPL5 39 7 has an estimated molecular weight of 43.2 kDa and an estimated pi of 7.4.

Comparison of amino acid sequence SEQ ID NO:43 with amino acid sequences reported in GenBank indicates that SEQ ID NO:43 showed the most homology, 28% identity with the protein encoded by a Drosophila melanogaster cDNA Accession CAA19845.2. Comparison of SEQ ID NO:45 with nucleic acid sequences reported in GenBank indicates that SEQ ID NO:45 showed the most homology, 40% with a Trichoplusia ni insect intestinal mucin IIM22, GenBank Accession #AF000606. Percent identity calculations were performed using the SeqLab software with default parameters.

A nucleic acid molecule comprising nucleotides 194 through 1354 of SEQ ID NO:42, encoding a predicted mature PL5 protein, was PCR amplified from the HMT RACE pool cDNA described above as follows. Sense primer PL5-FE, having nucleotide sequence 5' CGG GTA CCT TGG AGT CTC AAG AAC TAA TTC 3', which corresponds to nucleotides 194-215 of SEQ ID NO:42 and having a KpnI site indicated in bold, designated herein as SEQ ID NO:67, was used in conjunction with anti-sense primer PL5-RE, having nucleotide sequence 5' AGG AAT TCC ATA TAA CAC ACT CAC TAG GTA CAT GTA G which is complementary to nucleotides 194-215 of SEQ ID NO:42 and having an EcoRI site indicated in bold, designated herein as SEQ ID NO:68 under the following thermocycling conditions: one cycle of 940 for 30 seconds, 30 cycles of 940 for 30 seconds, 68° for 30 seconds, and 72' for 3 minutes, one cycle of 72° for 7 minutes. The products of this reaction were separated on a 1.5% agarose gel, and the appropriate band cut from the gel and 1^ purified using the QIAquick Gel Extraction Kit, available from Qiagen. The resulting N nucleic acid molecule, referred to herein as nCfPL5 1 6 1 has a coding strand designated SEQ ID NO:47 and a complementary strand designated SEQ ID NO:49. Translation 0 of SEQ ID NO:47 indicates that SEQ ID NO:47 encodes an approximately 381 amino acid protein, the amino acid sequence of which is designated SEQ ID NO:48, assuming a first codon at nucleotides 1 through 3 and a stop codon at nucleotides 1144 through 1146 of SEQ ID NO:47. The purified product was digested with KpnI and EcoRI and ligated into the vector pTrcHisB, available from Invitrogen, that had been digested with KpnI and EcoRI and treated with alkaline phosphatase. An individual clone was isolated and the DNA extracted and sequenced. DNA sequence analysis of the clone confirmed that nucleotides 194-1354 of SEQ ID NO:42, had been successfully ligated into the pTrcHisB expression vector in frame with the N-terminal T7 Tag epitope encoded by the vector. The recombinant protein, including the T7 Tag, is predicted to be 423 amino acids in length, have a molecular mass of 45.9 kDa and have a pI of 6.36.

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:

Claims (18)

1. An isolated nucleic acid molecule selected from the group consisting of a flea cDNA molecule and a flea RNA molecule, wherein said nucleic acid molecule is 00 C 5 selected from the group consisting of(a) a nucleic acid molecule at least 25 nucleotides in length that hybridizes with a nucleic acid molecule having a nucleic acid sequence Sselected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID C, NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID 0 10 NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ LD C NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32, and a nucleic acid molecule at least 35 nucleotides in length that hybridizes with a nucleic acid molecule having a nucleic acid sequence selected from the group consisting of SEQ ID NO:33, is SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:49; wherein said hybridization of(a) and (b) is performed under conditions comprising hybridizing in a solution comprising IX SSC in the absence of nucleic acid helix destabilizing compounds, at a temperature of 37C and washing in a solution comprising IX SSC in the absence of helix destabilizing compounds, at a temperature of 47°C.

2. The nucleic acid molecule of Claim 1, wherein said nucleic acid molecule has a nucleic acid sequence that is at least 70% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, 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:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:49, wherein percentage identity is determined N \Melboumc\CasesPatent\48000-48999\P48504 AU I\SpccS\P48504 AU I Specification 2007.9-27doc 27/09/07 using the Needleman-Wunsch algorithm available in a SeqLab software program, using SeqLab default parameters.

3. The nucleic acid molecule of Claim 1, wherein said nucleic acid molecule is selected from the group consisting of a nucleic acid molecule 00 C 5 comprising a nucleic acid sequence at least 25 nucleotides in length identical to a nucleotide portion of 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:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:13, SEQ C ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID CN NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32; and a nucleic acid molecule comprising a nucleic acid sequence at least nucleotides in length identical to a 35 nucleotide portion of a nucleic acid sequence selected from the group consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID is SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:49.

4. The nucleic acid molecule of Claim 1, wherein said nucleic acid molecule is selected from the group consisting of: a nucleic acid molecule that encodes a protein having an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:31, SEQ ID SEQ ID NO:43, and SEQ ID NO:48. A recombinant molecule comprising a nucleic acid molecule as set forth in Claim 1 operatively linked to a transcription control sequence.

6. A recombinant virus comprising a nucleic acid molecule as set forth in Claim 1.

7. A recombinant cell comprising a nucleic acid molecule as set forth in Claim 1.

8. A composition comprising an excipient and an isolated nucleic acid molecule of Claim 1.

9. A method to protect an animal from flea infestation comprising administering to said animal a composition of Claim 8. N \Melboumc\Cases\Patenr/48000-48999P485O4.AU I\Specis\P49S04 AU I Spcificalton 2001-9.27.do 27/09/07 I -76- The composition of Claim 8, further comprising a component selected Cfrom the group consisting of an adjuvant and a carrier.

11. A method to produce a protein encoded by an isolated nucleic acid molecule of Claim 1, said method comprising culturing a cell transformed with a 00 nucleic acid molecule encoding said protein.

12. The method of Claim 11, wherein said protein has an amino acid Ccr sequence selected from the group consisting of: SEQ ID NO:2, SEQ ID NO: 12, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:40, SEQ ID NO:43, and SEQ ID CN NO:48.

13. The method of Claim 11, wherein said nucleic acid molecule comprises C-K a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:19, SEQ rD NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:42, SEQ ID and SEQ ID NO:47.

14. An isolated nucleic acid molecule having a nucleic acid sequence selected from the group consisting of: a nucleic acid sequence comprising at least nucleotides identical in sequence to a 25 nucleotide portion of 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:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32; and a nucleic acid sequence comprising at least 35 nucleotides identical in sequence to a 35 nucleotide portion of a nucleic acid sequence selected from the group consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:49. An isolated protein selected from the group consisting of: a protein encoded by a nucleic acid molecule selected from the group consisting of(1) a nucleic N \Mcbomri\Cascs\Paten\4900OO48999\P48504 AU I\Spcis\P48504 AU, I Specificarion 2007.9-27doc 27/0907 -77- acid molecule at least 25 nucleotides in length that hybridizes with a nucleic acid C molecule having 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:5, SEQ ID NO:6, SEQ ID NO:7, V) SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ 00 N 5 ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:32, and a nucleic acid molecule at least 35 nucleotides in length that hybridizes CI with a nucleic acid molecule having a nucleic acid sequence selected from the group O 10 consisting of SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID N NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:49, wherein said hybridization of(1) and is performed under conditions comprising (i) hybridizing in a solution comprising IX SSC in the absence of nucleic acid helix destabilizing compounds, at a temperature of 37 0 C and (ii) washing in a solution comprising IX SSC in the absence of helix destabilizing compounds, at a temperature of 47°C; and an isolated protein, comprising at least 10 amino acids identical in sequence to a 10 amino acid portion of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:40, SEQ ID NO:43, and SEQ ID NO:48.

16. The protein of Claim 15, wherein said protein is encoded by a nucleic acid molecule having a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, and SEQ ID NO:47.

17. The protein of Claim 15, wherein said protein comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:40, SEQ ID NO:43, and SEQ ID NO:48. N \Mclbor,,CaCSe\PlenI \48000-4S999\P48504 AU I\Spccis\P48504 AU I Specification 2007.9-27 doc 27/09107 -78-

18. A composition comprising an excipient and an isolated protein of Claim

19. A method to protect an animal from flea infestation comprising administering to said animal a composition of Claim 18. 00 l 5 20. An isolated antibody that selectively binds to a protein as set forth in Claim €r 21. A composition comprising an excipient and an isolated antibody of Claim C1 22. A method to protect an animal from flea infestation comprising administering to said animal a composition of Claim 21. C 23. A method to detect an inhibitor of flea peritrophin activity, said method comprising contacting an isolated flea peritrophin protein of Claim 15, with a putative inhibitory compound under conditions in which, in the absence of said compound, said protein has flea peritrophin protein activity, and determining if said putative inhibitory compound inhibits flea peritrophin protein activity.

24. The method of Claim 23, wherein said flea peritrophin protein is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, and SEQ ID NO:47. The method of Claim 23, wherein said flea peritrophin protein has an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:40, SEQ ID NO:43, and SEQ ID NO:48. N \Melboume\Cases\Paten\4800O.48999\P48504 AU I/SpccisP48504 AU I Sptcification 2007-9-27 doc 27/09107 SEQUENCE LISTING <110> Gaines, Patrick J. Wisnewski, Nancy <120> FLEA PERITROPHIN NUCLEIC ACID MOLECULES, PROTEINS AND USES THEREOF <130> FC-6--C2-PCT <140> not yet assigned <141> 2001-10-11 <150> 09/686,583 <151> 2000-10-11 <150> 09/543,668 <151> 2000-04-07 <150> 60/128,704 <151> 1999-04-09 <160> 68 <170> Patentln Ver. 2.1 <210> 1 <211> 1096 <212> DNA <213> Ctenocephalides felis <220> <221> CDS <222> (821) <400> 1 tcaca atg aag ttc tta gga gct tta ttg gtt gca gtg ttt gcc ttg ggt Met Lys Phe Leu Gly Ala Leu Leu Val Ala Val Phe Ala Leu Gly 1 5 10 gct gtg gct gct Ala Val Ala Ala gac Asp agg aat tcg ccc Arg Asn Ser Pro ac a Thr 25 tat gtc cgc ggt Tyr Val Arg Gly ttc cca Phe Pro gtg gga aga Val Gly Arg tgt act aat Cys Thr Asn aga gca cga aca aca ttt ggc aat gaa Arg Ala Arg Thr Thr Phe Giy Asn Giu gaa ata aag Glu Ile Lys tct act ttg Ser Thr Leu aag cag ttg gga Lys Gin Leu Giy ttt tgt cac gat Phe Cys His Asp aag ttg Lys Leu tgc gct gga caa gaa acc cca att aca Cys Ala Gly Gin Glu Thr Pro Ile Thr atc aat tgc aga Ile Asn Cys Arg gac Asp tca aat tcc gat Ser Asn Ser Asp cca ttt tgt Pro Phe Cys gta gat Val Asp gat atg tgc tca Asp Met Cys Ser aaa cct ggg gaa aac tgt aag acg gca gaa act aca tgc gcc gtt gta Lys Pro Gly Glu Asn Cys Lys AlGlThTr 00 Ala Glu Thr Thr gga Gly gat Asp cat His gtc Val 160 cca Pro aaa Lys tgc Cys tgt Cys aaa Lys 240 cga Arg tat Tyr ggt Gly tct Ser 145 atg Met tca Ser tgt Cys aga Arg aaa. Lys 225 cat His tgc Cys cag Gin aaa Lys 130 aaa Lys aaa Lys att Ile gaa Giu act Thr 210 aaa Lys tat Tyr ata Ile cca Pro 115 ggt Giy aat Asn tgc Cys tat Tyr gac Asp 195 gca Ala tat Tyr gat Asp ccc Pro gat Asp cag Gin atg Met aca Thr gc L Ala 180 gac Asp tgc Cys tat Tyr tgt Cys acg Thr 260 ccg aaa gac Pro Lys Asp gtt ttc gaa Val Phe Glu 135 tgt aaa aag Cys Lys Lys 150 aat ccc aat Asn Pro Asn 165 tgg tgc aat Trp Cys Asn gtc aac gaa Val Asn Giu aaa agt gaa Lys Ser Giu 215 caa tgt ttc Gin Cys Phe 230 cca aat ggc Pro Asn Giy 245 cca ccc ggc Pro Pro Gly Lgc Cys 120 tgc Cys aaa Lys tc t Ser gac Asp tgg Trp 200 aac Asn ttg Leu ttg Leu gaa Glu aca aga Lac Thr Arg Tyr cca cct aac Pro Pro Asn *tcg tca gaa Ser Ser Glu 155 ttt ata acc Phe Ile Thr 170 aaa ttg caa Lys Leu Gin 185 ttt gac cca Phe Asp Pro gtt ttt Lcc Val Phe Ser gtt aac aac Val Asn Asn 235 cac ttt gat His Phe Asp 250 gaa tgc aaa Glu Cys Lys 265 Cys tta Leu tat Tyr 140 gc L Ala tat Tyr ccg Pro aaa Lys gat Asp 220 aaa Lys aaa Lys agt ttc Phe 125 gta Val gat Asp gca Ala atc Ile tct Ser 205 cga Arg tgg Trp acg Thr gag tgc Cys tat Tyr tgc Cys ccg Pro gta Val 190 ttLc Phe aga Arg caa Gin gag Glu att aaa L~ys gaL Asp acc Thr gac Asp 175 ctg Leu tcg Ser gaL Asp ata Ile ttg Leu 255 gc L Ala Val Val 110 386 434 482 530 578 626 674 722 770 818 871 931 991 Ser Giu Ile Ala aag Laaggcttaa accaggaaaa caatcttgaa tagactaatt aggattcaaa Lys ttatcataaa. gtagtcaatt aatataataa atacacaaat gatctgtgca attaaatata. aaaaatatgt ttaaaaatta aaatgtataa aattgtattt tatgtaagga gcacaaacaa aatgtcctta actatagtaa tttctgatta tttaaaatat ataaatatag aagctttatg 1051 aaattacatg tatcttttta ataaaaataa atcgtttggg ccgtt 1096 <210> 2 <211> 272 <212> PRT <213> Ctenocephaiides felis <400> 2 Met Lys Phe Leu Gly Ala Leu Leu Vai Ala Val Phe Ala Leu Gly Ala 10 Val Ala Ala Asp Arg Asn Ser Pro Thr Tyr Val Arg Gly Phe Pro Val 25 00 Gly Arg Ser Arg Ala Arg Thr Thr Phe Gly Asn Giu Giu Ile Ly's Cys 40 Thr Asn Lys Gin Leu Giy Thr Phe Cys His Asp Cys Ser Thr Leu Lys 55 Leu Cys Ala Gly Gin Giu Thr Pro Ile Thr Thr Ile Asn Cys Arg Asp 70 75 Ser Asn Ser Asp Ala Pro Phe Cys Val Asp Asp Met Cys Ser Ser Lys 90 Pro Gly Giu Asn Cys Lys Thr Ala Giu Thr Thr Cys Ala Val Val Gly 100 105 110 Tyr Gin Pro Asp Pro Lys Asp Cys Thr Arg Tyr Leu Phe Cys Lys Asp 115 120 125 Gly Lys Gly Gin Val Phe Giu Cys Pro Pro Asn Tyr Val Tyr Asp His 130 135 140 Ser Lys Asn Met Cys Ly's Lys Lys Ser Ser Giu Ala Asp Cys Thr Val 145 150 155 160 Met Lys Cys Thr Asn Pro Asn Ser Phe Ile Thr Tyr Ala Pro Asp Pro 165 170 175 Ser Ile Tyr Ala Trp Cx's Asn Asp Lys Leu Gin Pro Ile Val Leu Lys 180 185 190 Cys Giu Asp Asp Val Asn Giu Trp, Phe Asp Pro Lys Ser Phe Ser Cys 195 200 205 Arg Thr Ala Cys Lys Ser Giu Asn Val Phe Ser Asp Arg Arg Asp Cys 210 215 220 Lys Lys Tyr Tyr Gin Cys Phe Leu Val Asn Asn Lys Trp Gin Ile Lys 225 230 235 240 His Tyr Asp Cys Pro Asn Gly Leu His Phe Asp Lys Thr Glu Leu Arg 245 250 255 Cys Ile Pro Thr Pro Pro Gly Giu Giu Cys Lys Ser Glu Ile Ala Lys 260 265 270 <210> 3 <211>, 1096 <212> DNA <213> Ctenocephalides felis <400> 3 00 aacggcccaa acgatt tttatatatt ttaaat acataaaata caattt agatcatttg tgtatt gtctattcaa gattgt tcgccgggtg gcgtgg caatcataat gtttta caatctcttc gatcgg tttgggtcaa accatt tcattgcacc aagcat gtgcatttca tgacgg tgatcatata catagt aagtatcttg tgcagt gccgtcttac agtttt tcggaatttg agtctc ttcaaagtag aacaat tcattgccaa atgttg 1020 ggcgaattcc tgtcag 1080 aagaacttca ttgtga 1096 tatt aatc tata .tatt .tttc gtat .tttg aaaa cgtt aaat tgca tagg cttt cccc tgca cgtg ttcg tttattaaaa agaaattact cattttaatt atattaattg ctggtttaag gcatcgcaac ccatttgttg aacgttttca gacgtcgtct tgatgggtcc atcagcttct tgggcattcg cggatctggc aggttttgat attgattgtt acaaaatgtt tgctctggat agatacatgt atagttaagg tttaaacata actactttat ccttacttag tccgttttat ttaaccaaga cttttgcatg tcacatttca ggtgcatagg gacgatttct aaaacctgac tgatatccta gagcacatat gtaattgggg cccaactgct cttcccactg aatttcataa acattttgtt ttttttatat gataatttga caatctcact caaagtgcaa a acat tgata cagttctgca gtacgatcgg ttataaaaga ttttacacat ctttaccatc caacggcgca catctacaca tttcttgtcc tattagtaca ggaaaccgcg agcttctata tgtgctcctt ttaattgcac atcctaatta tttgcattct gccatttgga atatttttta cgagaaagat ttgcaattitg attgggattt atttttagaa tttgcagaat tgtagtttct aaatggagca agcgcacaac ctttatttct gacatatgtg 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 cagc cacagcaccc aaggcaaaca ctgcaaccaa taaagctcct <210> 4 <211> 816 <212> DNA <213> Ctenocephalides felis <400> 4 atgaagttct aggaattcgc tttggcaatg tctactttga tcaaattccg tgtaagacgg acaagatac t gtatatgatc atgaaatgca tggtgcaatg tttgacccaa cgaagagatt cattatgatt ccacccggcg taggagcttt ccacatatgt aagaaataaa agttgtgcgc atgctccatt cagaaactac tattctgcaa at tctaaaaa caaatcccaa acaaattgca aatctttctc gtaaaaaata gtccaaatgg aagaatgcaa attggttgca ccgcggtttc gtgtactaat tggacaagaa ttgtgtagat atgcgccgtt agatggtaaa tatgtgtaaa ttcttttata accgatcgta gtgcagaact ttatcaatgt cttgcacttt aagtgagatt gtgtttgcct ccagtgggaa aagcagttgg accccaatta gatatgtgct gtaggatatc ggtcaggttt aagaaatcgt acctatgcac c tgaaatgtg gcatgcaaaa ttcttggtta gataaaacgg gctaag tgggtgctgt gatccagagc gaacattttg caacaatcaa catcaaaacc agccagatcc tcgaatgccc cagaagctga cggacccatc aagacgacgt gtgaaaacgt acaacaaatg agttgcgatg ggctgctgac acgaacaaca tcacgattgt ttgcagagac tggggaaaac gaaagactgc acctaactat ttgcaccgtc aatttatgct caacgaatgg tttttccqat gcaaataaaa catacccacg 120 180 240 300 360 420 480 540 600 660 720 780 816 <210> <211> 816 <212> DNA <213> Ctenocephalides felis <400> cttagcaatc tttatcaaag caagaaacat gcatgcagtt tttcagtacg ataggttata tttcttttta c tgaccttta tcctacaacg tcacttttgc tgcaagccat tgataatatt ctgcacgaga atcggttgca aaagaattgg cacatatttt ccatctttgc gcgcatgtag attcttcgcc ttggacaatc ttttacaatc aagattttgg atttgtcatt gatttgtgca tagaatgatc agaataagta tttctgccgt gggtggcgtg ataatgtttt tcttcgatcg gtcaaaccat gcaccaagca tttcatgacg atatacatag tcttgtgcag cttacagttt ggtatgcatc atttgccatt gaaaaaacgt tcgttgacgt taaattgatg gtgcaatcag ttaggtgggc tctttcggat tccccaggtt gcaac tccgt tgttgttaac tttcactttt cgtcttcaca ggtccggtgc cttctgacga attcgaaaac ctggctgata ttgatgagca catatcatct acacaaaatg tggggtttct tgtccagcgc ctgcttatta gtacacttta cactgggaaa ccgcggacat aaacactgca accaataaag gagcatcgga acaacttcaa tttcttcatt atgtgggcga ctcctaagaa atttgagtct ctgcaattga ttgttgtaat 600 agtagaacaa tcgtgacaaa atgttcccaa 660 gccaaatgtt gttcgtgctc tggatcttcc 720 attcctgtca gcagccacag cacccaaggc 780 cttcat 816 <210> 6 <211> 445 <212> DNA <213> Ctenocephalides fells <400> 6 gcaaacaaca tagaaattgg tcaaattaaa ttccagacat attattcatg attttgatcg acaaagttca taataaaata aggtccgctg atcattaccg atgcgtgaaa ttgcgatgaa cgtcacgatt agaaagatta atctttaatt. gtttattggc atgcaagatc aaacatttta ggaaattgcg gtaggacctt ggaaaagaac cggtgtgtca attatttaga aattt cacacgattg attgtaataa aaaatagcac tggtgcaaga ctgaacattt gaggatcttg agaatttgaa tcacgcatat aggtgcttat agaaattcct tccaaacgat tacgtgcaat ttaacaaata aatgtatatt tacacatgtc ttcaatacag cttcctgagc tgccgcaagt aaaggggcgt ttgttatata taatgttttt 120 180 240 300 360 420 445 <210> 7 <211> 1279 <2 12> DNA <213> Ctenocephalides felis <400> 7 gattagctgc catgtttctc attgtcagcg ggggttacaa cgtgtaaacg atagaaatat tatatccttt gcgtaagaga gtgaaacaac ccacgacgtc ctgccacaac caccaaccga aatcttccac caaagcctac aacaaggtcc ttggatcatt taaaatgcgt 1020 acatttgcga 1080 catgcgtcac 1140 atcgagaaag 1200 ttcaatcttt 1260 aatagtttat 1279 <210> 8 <211> 1279 ccactatagg ggaaggttct ttatcatgaa ggcttataat cttacaattt atattatagg attatatcgg tgaaaccacc cacgacgtct tgccacaaca accaaccgaa atcttccacg gtctggtgaa ttctacggaa gctgatgcaa accgaaacat gaaaggaaat gctaaagcgg ataacttgca tgtaaaactg gttatagaaa cattgtgata tgcaccgaaa tgtgatgaga acgacgcctg gccacaacat ccaaccgaat tcttccacgt tc tagtgaaa ac aac cacga actcccgcaa gatccacacg tttaattgta tgcgaaaata ccgccgggtg ccggtccagg caaatgaatc ataattgtag ctataggaga aaaccttgtg gtgagatata ccacaacgcc caaccgaatc cttccacgtc ctagtgaaac caaccacgac cgtctgccac caaaaccacc attgtcacgc ataaaggtac gcacagaaat gtgttctgca agtttttcct atctaagcct cctgaacatg tgaaaatgct gttcaatagc tgatgcagtg aaccgaatct ttccacgtct tagtgaaaca aaccacgacg gtctgccaca aacaccaacc gcaagaaata atattacaca ttatttcaat tcctcttcct gatccaagcc gatccgtatg gtcgagtgtg aatcatcaat tggccgagca aacaaaatat cagagagttt tccacgtcta agtgaaacaa accacgacgt tctgccacaa acaccaaccg gaaccttcca ccatgcaaac tgtctagaaa acagtcaaat gagcttccag 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 tgaagtagga cctttggtgc aagatccaaa cgattgccgc aagtattatt gattggaaaa gaacctgaac attttacgtg caataaaggg gcgtattttg attacggtgt gtcagaggat cttgttaaca aatattgtta tataacaaag aattattatt tagaagaatt tgaaaatgta tatttaatgt tttttaataa tggcaattt 00 <212> DNA <21.3> Ctenocephalides felis <400> 8 aaat tgccaa ataataatta caccgtaatc tttccaatcg cctacttcat tttcctttca tgtttcggta tgcatcagcg tccgtagaag tcaccagacg gtggaagatt tcggttggtg gttgtggcag gacgtcgtgg gtggtttcat cgatataata ctataatata 1020 aattgtaagc 1080 ttataagcct 1140 tcatgataac 1200 gaaccttccg 1260 ctatagtggg 1279 taaac tat tt aaga ttgaac tttctcgatc tgacgcatga cgcaaatgtc cgcattttaa atgatccaat gaccttgttg taggctttgt tggaagattc cggt tggtgt ttgtggcaga acgtcgtggt ttgtttcact ctcttacgca aaggatataa tatttctatt tattaaaaaa tttgttatat aaaatacgcc ataatacttg tggaagctca tttgactgta ttctagacat tttgcatggt ggaaggttcg ggttggtgtt tgtggcagac cgtcgtggtt tgtttcacta agacgtggaa aactctctgc tattttgttg gctcg-gccaa cattaaatat aacaatattt cctttattgc cggcaatcgt ggaagaggaa ttgaaataag gtgtaatatg atttcttgcg gttggtgttg gtggcagacg gtcgtggttg gtttcactag gacgtggaag gattcggttg actgcatcat ctattgaacc gcattttcat acattttcaa gttaacaaga acgtaaaatg ttggatcttg tttctgtgct cacctttatt cgtgacaatc gtggttttgt tggcagacgt tcgtggttgt tttcactaga acgtggaaga attcggttga gcgttgtggc atatctcact acaaggtttt ctcctatagt attcttctaa tcctctgaca ttcaggttct caccaaaggt attttcgcaa acaat taaaa gtgtggatct tgcgggagtt cgtggttgtt ttcactagac cgtggaagat ttcggttggt tgt tgtggca aggcgtcgtg ctcatcacac ttcggtgcac atcacaatga 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 gtttacacga ttgatgattc atgttcaggc tacaattatt ttctataaca tgtaaccccc acactcgaca ggcttagatg attcatttgc agttttacat gctgacaatc atacggatca ggaaaaactc ctggaccggt gcaagttata agaaacatgg gcttggatct gcagaacacc acccggcggc cgctttagcc cagctaatc <210> 9 <211> 279 <212> DNA <213> Ctenocephalides fells <400> 9 caatgatcaa ttgataatcg actgcagcgt caagtccatg caagcccatg gtcagttata gcagactaac cacagctttt tagttctggt tttctcggaa attgtggcca acttttggct aactgctctg agattggtgt tgtggtagag gacctacggg gaagtttgca gtacctgggc ggttctataa cttgcaccg attcgtagcc ttgcaatcga tgatggtttt ttttgtctca 120 tgaattcaac accgtgtcta 180 gggtagatgt tctgcagatc 240 279 <210> <211> 279 <212> DNA <213> Ctenocephalides felis <400> cggtgcaagt tatagaacct cccaggtact gcaaacttca ccgtaggtcc tctaccacaa caccaatctc agagcagttg gccaaaagtt ggccacaatt tccgagaaac atgggcttgg atctgcagaa catctacccg ccagaactac atggacttgt agacacggtg ttgaattcac 120 aaagctgtga cgctgcagtt gagacaaaaa aaaccatcaa 180 ttagtctgcc gattatcaat cgattgcaag gctacgaata 240 ataactgact tgatcattg 279 <210> 11 <211> 1465 <212> DNA <213> Ctenocephaiides felis <220> <221> CDS <222> (3)..(1361) <400> 11 ca atg atc aag tca gtt ata att gtg gcc aac ttt tgg cta ttc gta Met Ile Lys Ser Val Ile Ile Val Ala Asn Phe Trp Leu Phe Vai goc ttg caa tog Ala Leu Gin Ser gat aat cgg cag Asp Asn Arg Gin act aac aac tgc Thr Asn Asn Cys tgc agc gtc aca Cys Ser Val Thr ggt gtt gat Gly Val Asp ggt aga gga Gly Arg Gly ggt Gly ttt ttt tgt ctc Phe Phe Cys Leu tct gag att Ser Giu Ile got ttt tgt Ala Phe Cys agt cca tgt Ser Pro Cys cct acg ggt gaa Pro Thr Gly Giu aac aco gtg tot Asn Thr Val Ser aca Thr agt tot Ser Ser ggt gaa gtt tgo Giy Glu Val Cys agt Ser 70 aco tgg gcg ggt Thr Trp Ala Gly aga A-rg tgt. tot gca gat Cys Ser Ala Asp coa Pro ago cca tgt ttc Ser Pro Cys Phe tog Ser gaa ggt tot ata Glu Giy Ser Ile tgc acc ggt oca Cys Thr Gly Pro 239 287 335 gtt ttt cot gat Val Phe Pro Asp tat gat tgt cag Tyr Asp Cys Gin cgt Arg 105 tat oat gaa tgt Tyr His Giu Cys aaa act Lys Thr 110 gca aat gaa Ala Asn Giu aat gtt ata Asn Val Ile 130 toa Ser 115 tot aag cot gtc Ser Lys Pro Vai tgt ggg ggt tao Cys Giy Gly Tyr aag got tat Lys Ala Tyr 125 oaa tog tgt Gin Ser Cys gaa aat aat tgt Giu Asn Asn Cys ctg aac atg aat Leu Asn Met Asn oat His 140 aaa ogo Lys Arg 145 tta caa ttt cat Leu Gin Phe His tgt Cys 150 gat act ata gga Asp Thr Ile Gly gaa aat got tgg Giu Asn Ala Trp cog Pro 160 ago aat aga aat Ser Asn Arg Asn tat tat agg tgc Tyr Tyr Arg Cys ac c Thr 170 gaa aaa acc ttg Giu Lys Thr Leu 479 527 575 ttc aat ago aac Phe Asn Ser Asn aaa Lys 180 ata tta tat cot Ile Leu Tyr Pro tta tat cgg tgt Leu Tyr Arg Cys gat gag Asp Glu 190 agt gag ata Ser Glu TIP tat gat Tyr Asp 195 gca gtg oag Ala Val Gin aga Arg 200 gtt tgc gta aga Vai Cys Val Arg gat gaa aco Asp Glu Thr 205 8 aco acg acg oct gcc aca acg cca acc gaa tot tcc acg tct agt gaa Thr Thr Thr Pro Ala Thr Thr Pro Thr Glu Ser Ser Thr Ser Ser Giu 00 210 220 aca act Thr Thr 225 gaa aca Glu Thr 240 agt gaa Ser Glu tot agt Sex Ser acg tot Thr Ser tcc acg Ser Thr 305 Oct too 1007 Pro Ser 320 caa gaa 1055 Gin Giu gat tgt 1103 Asp Cys cat ttt 1151 His Phe tgo gtg 1199 Cys Val 385 ott cca 1247 Leu Pro 400 gat tgc 1295 acg Thr act Thr aca Thr gaa Glu agt Ser 290 tct S er aca Thr ata Ile cac His aat Asn 370 aaa Lys gac Asp cgc acg Thr acg Thr act Thr aca Thr 275 gaa Glu ggt Gly aag Lys cca Pro gca Ala 355 tgt Cys gga Gly att Ile aagI tot Ser acg Thr aog Thr 260 acc Thr aca Thr gaa Giu ct Pro tgc Cys 340 tat T'yr aa t Asn aat ksn :gc tat gc Ala tct Ser 245 aog Thr aog Thr act Thr aca Thr act Thr 325 aaa Lys tac Tyr aaa Lys tgc Cys gat Asp 405 tat aca Thr 230 gcc Ala tot Ser acg Thr aog Thr ac Thr 310 tot Ser caa Gin aoa Thr ggt Gly gaa. Glu 390 gaa G1u tca. aca Thr aca Thr gczo Ala tot Ser acg Thr 295 acg Thr aeg Thr o aa Gin tgt Cys gct Ala 375 aat Asn gta Val tgc Cys tca Ser aca Thr aca Thr gc Ala 280 tct S ex acg Thr gaa Glu ggt Gly cta. Leu 360 tat Tyr agc Ser gga Gly gtc act Thr ca Pro ata Thr 265 aoa Thr gcc Ala tot Ser act Thr ttg Pro 345 gaa Glu t tt Phe aca Thr ct Pro acg gaa Giu act Thr 250 oca Pro aca Thr aca Thr gco Ala ccc Pro 330 otg Leu at t Ile aat Asn gaa Giu ttg Leu 410 att tt Ser 235 gaa Glu act Thr oca Pro aca Thr aca Thr 315 gca Ala atg Met gga Gly aca Thr att Ile 395 gtg Val gga too Ser tot Ser gaa Giu at o Thr ca Pro 300 aca Thr aca Thr o aa Gln tta Ser gto Val 380 oct Pro o aa Gln ~aaa acg Thr tcc Ser tt Ser gaa Giu 285 acc Thr cca Pro aaa Lys gat Asp tta Leu 365 aaa Lys ctt Leu gat Asp gaa tct Ser a og Thr too Ser 270 tot Ser gaa Giu act Thr oca Pro oca Pro 350 tog Pro tta Leu cot Pro coa Pro ot agt Ser tot Ser 255 acg Thr tot Sex tt Ser gaa Glu ccg Pro 335 cat His aaa Lys aaa Lys gag Glu aac Asn 415 gaa 67 1 719 767 815 863 911 959 Asp Cys Arg Lys Tyr Tyr Ser Va h i l y l r l Val Thr Ile Gly Lys Glu Pro Glu 00 420 425 430 cat ttt acg tgc aat aaa ggg gcg tat ttt gat cga gaa aga tta cgg 1343 His Phe Thr Cys Asn Lys Gly Ala Tyr Phe Asp Arg Giu Axg Leu Arg 435 440 445 tgt gtc aga gga tct tgt taacaaatat tgttatataa caaagttcaa 1391 Cx's Val Arg Gly Ser Cys 450 tctttaatta ttatttagaa gaatttgaaa atgtatattt aatgtttttt aataaaatag 1451 tttattggca attt 1465 <210> 12 <211> 453 <212> PRT <213> Ctenocephaiides felis <400> 12 Met Ile Lys Ser Val Ile Ile Val Ala Asn Phe Trp Leu Phe Val 1 Leu Val Arg S er Ser Phe Asn Val Arg 145 Ser Gin Asp Gly Gly Pro Pro Giu Ile 130 Leu Asn Ser Gly Pro Glu Cys Asp Ser 115 Giu Gin Arg Ile Phe Thr Val Phe Pro 100 Ser Asn Phe Asn 5 Asp Phe Gly Cys Ser Tyr Lys Asn His Ile 165 Asn Cys Glu Ser 70 Glu Asp Pro Cys Cys 150 Tyr Arg Leu Phe 55 Thr Gly Cys Val Ser 135 Asp Tyr Gin Asn 40 Asn Trp Ser Gin Giu 120 Leu Thr Arg Thr 25 Cys Thr Ala Ile Arg 105 Cys Asn Ile Cys 10 Asn S er Val Gly Thr 90 Tyr Gly Met Gly Thr Asn Val Ser Arg 75 Cys His Giy Asn Asp 155 Glu Cys Thr Thr Cys Thr Giu Tyr His 140 Giu Ly's Ser Giu Ala Phe Ser Pro Ser Ala Gly Pro Cx's Lys 110 Lys Ala 125 Gin Ser Asn Ala Thr Leu Ile Cx's Cx's Asp Gix' Thr Tyr Cys Trp Trp, Ala Gly Gly Ser Pro Val Ala Asn Ly's Pro 160 Phe Asn Ser Asn Lys Ile Leu Tyr Pro Leu Leu Tyr Arg Cys Asp Giu Ser 180 185 190 Arg Val 00 Glu Ile Tyr 195 Asp Ala Val Gin Cys Val Arg Asp Giu Thr Thr 205 200 Thr Thr 225 Thr Clu Ser Ser Thr 305 Ser Giu Cys Phe Val 385 Pro Cys Phe Val Thr 210 Thr Thr Thr Glu Ser 290 Ser Thx Ile His Asn 370 Lys Asp Arg Thr Arg 450 Pro Thr Thr Thr Thr 275 Glu Gly Ly's Pro Ala 355 Cys Gly Ile Lys Cx's 435 Gly Al a Ser Thr Thr 260 Thr Thr Glu Pro Cys 340 Tyr Asn Asn Cys Tyr 420 Asn Ser Thr Ala Ser 245 Thr Thr Thr Thr Thr 325 Lys Tyr Lys Cys Asp 405 Tyr Lys cx's Thr Thr 230 Ala Ser Thr Thr Thr 310 Ser Gin Thr Gly Giu 390 Giu Ser Gly Pro 215 Thr Thr Ala Ser Thr 295 Thr Thr Gin Cys Ala 375 Asn Val Cx's Ala Thr Ser Thr Thr Ala 280 Ser Thr Glu Gly Leu 360 Tyr Ser Gly Val Tyr 440 Glu Thr Pro Thr 265 Thr Ala Ser Thr Pro 345 Glu Phe Thr Pro Thr 425 Phe Ser G lu Thr 250 Pro Thr Thr Ala Pro 330 Leu Ile Asn Glu Leu 410 Ile Asp Ser Ser 235 Giu Thr Pro Thr Thr 315 Ala Met Gly Thr Ile 395 Val Glx' Arg Thr 220 Ser Ser Glu Thr Pro 300 Thr Thr Gin Ser Val 380 Pro Gin Ly's Glu *Ser Thr Ser Ser Glu 285 Thr Pro Lx's Asp Leu 365 Lx's Leu Asp Giu Arg 445 S er Ser Thr Ser 270 Ser Glu Thr Pro Pro 350 Pro Leu Pro Pro Pro 430 Leu Giu Ser Ser 255 Thr Ser Ser Glu Pro 335 His Lys Lx's Glu Asn 415 Giu Arg Thr Glu 240 Ser Ser Thr Ser Pro 320 Gin Asp His Cys Leu 400 Asp His Cx's <210> 13 <211> 1465 <212> DNA <213> Ctenocephaiides felis <400> 13 aaattgccaa taaactattt tattaaaaaa cattaaatat acattttcaa attcttctaa ataataatta aagattgaac tttgttatat aacaatattt gttaacaaga tcctctgaca 120 00 caccgtaatc tttccaatcg cctacttcat tttcctttca tgtttcggta tgcatcagcg tccgtagaag tcaccagacg gtggaagatt tcggttggtg gttgtggcag gacgtcgtgg gtggt ttca t cgatataata ctataatata 1020 aattgtaagc 1080 ttataagcct 1140 tcatgataac 1200 gaaccttccg 1260 acttcaccag 1320 ccacaaaaag 1380 caglttgttag 1440 acaat tataa 1465 t t c tcga tc tgacgcatga cgcaaatgtc atgatccaat gaccttgttg taggctttgt tggaagattc cggttggtgt ttgtggcaga acgtcgtggt ttgtttcact ctcttacgca aaggatataa tatttctatt aaaatacgcc ataatacttg tggaagctca tttgactgta ttctagacat tttgcatggt ggaaggttcg ggttggtgt t tgtggcagac Cgtcgtggtt tgtttcacta agacgtggaa aactctctgc tattttgttg gctcggccaa cctttattgc acgtaaaatg ttcaggttct 180 cggcaatcgt ggaagaggaa ttgaaataag gtgtaatatg atttcttgcg gttggtgt tg gtggcagacg gtcgtggttg gtttcactag gacgtggaag gattcggttg actgcatcat ctattgaacc gcattttcat ttggatcttg tttctgtgct cacctttatt cgtgacaatc gtggttttgt tggcagacgt tcgtggttgt tttcactaga acgtggaaga attcggttga gcgttgtggc atatctcact acaaggtttt ctcctatagt caccaaaggt attttcgcaa acaattaaaa gtgtggatct tgcgggagt t cgtggttgtt ttcactagac cgtggaagat ttcggttggt tgttgtggca aggcgtcgtg ctcatcacac ttcggtgcac atcacaatga 240 300 360 420 480 540 600 660 720 780 840 900 960 gtttacacga ttgatgattc atgttcaggc tacaattatt ttctataaca tgtaaccccc acactcgaca 'ggcttagatg attcatttgc agttttacat gctgacaatc atacggatca ggaaaaactc ctggaccggt gcaagttata agaaacatgg gcttggatct gcagaacatc tacccgccca ggtactgcaa aactacatgg acttgtagac acggtgttga attcacccgt aggtcctcta ctgtgacgct gcagttgaga caaaaaaaac catcaa~cacc aatctcagag tctgccgatt atcaatcgat tgcaaggcta cgaatagcca aaagttggcc ctgacttgat cattg <210> 14 <211> 1359 <212> DNA <213> Ctenocephalides felis <400> 14 atgatcaagt gataatcggc tgcagcgtca agtccatgta agcccatgtt tatgattgtc tgtgggggtt caatcgtgta agcaatiagaa atattatatc gtttgcgtaa tctagtgaaa acaaccacga acgtctgcca acaacaccaa accgaatctt tccacaaagc 1020 cagttataat agactaacaa cagctttttg gttctggtga tctcggaagg agcgttatca acaaggctta aacgcttaca atatatatta ctttattata gagatgaaac caaccacgac cgtctgccac caacaccaac ccgaatcttc ccacgtctgg ctacttctac tgtggccaac ctgctctgag tggtagagga agtttgcagt ttctataact tgaatgtaaa taatgttata atttcattgt taggtgcacc tcggtgtgat caccacgacg gtctgccaca aacaccaacc cgaatcttcc cacgtctagt tgaaacaacc ggaaactccc ttttggctat attggtgttg cctacgggtg acc tgggcgg tgcaccggtc actgcaaatg gaaaataatt gatactatag gaaaaaacc t gagagtgaga cctgccacaa acatcaaccg gaat ct tcc a acgtctagtg gaaacaacca acgacgtctg gcaacaaaac tcgtagcctt atggtttttt aattcaacac gtagatgttc caggagtttt aatcatctaa gtagcctgaa gagatgaaaa tgtggttcaa tatatgatgc cgccaaccga aatcttccac cgtctagtga aaacaaccac cgacgtctgc cc ac aacac c caCCgCaaga gcaatcgatt ttgtctcaac Cgtgtctaca tgcagatcca tcctgatccg gcctgtcgag catgaatcat tgcttggccg tagcaacaaa agtgcagaga atcttccacg gtctagtgaa aacaaccacg gacgtctgcc cacaacacca aaccgaacct aataccatgc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 aaacaacaag gtccgctgat gcaagatcca cacgattgtc acgcatatta cacatgtcta 1080 00 gaaattggat cattaccgaa acattttaat 1140 aaattaaaat gcgtgaaagg aaattgcgaa 1200 ccagacattt gcgatgaagt aggacctttg 1260 tattcatgcg tcacgattgg aaaagaacct 1320 tttgatcgag aaagattacg gtgtgtcaga 1359 <210> <211> 1359 <212> DNA <213> Ctenocephalides felis tgtaataaag gtgcttattt caatacagtc aatagcacag aaattcctct tcctgagctt gtgcaagatc caaacgattg ccgcaagtat gaacatttta Cgtgcaataa aggggcgtat ggatcttgt <400> acaagatcct aaaatgttca atcttgcacc tgtgctattt tttattacaa acaatcgtgt ttttgttgcg agacgtcgtg ggttgtttca actagacgtg ggaagattcg ggttgatgtt tgtggcaggc ctcactctca ggttttttcg tatagtatca attattttct 1020 atttgcagtt 1080 accggtgcaa 1140 cgcccaggta 1200 acccgtaggt 1260 aacaccaatc 1320 tagccaaaag 1359 ctgacacacc ggttcttttc aaaggtccta tcgcaatttc ttaaaatgtt ggatcttgca ggagtttccg gttgtttcac ctagacgtgg gaagattcgg gttggtgttg gtggcagacg gtcgtggtgg tcacaccgat gtgcacctat caatgaaatt ataacat tat gtaatctttc caatcgtgac cttcatcgca ctttcacgca tcggtaatga tcagcggacc tagaagtagg cagacgtgga aagattcggt ttggtgttgt tggcagacgt tcgtggttgt tttcatctct ataataaagg aatatatatt gtaagcgttt aagccttgta tcgatcaaaa gcatgaataa aatgtctgga ttttaatttg tccaatttct ttgttgtttg ctttgtggaa agattcggtt tggtgt tgtg ggcagacgtc cgtggttgtt ttcactagac tacgcaaact atataatatt tctattgctc acacgattga acccccacac tacgcccctt tacttgcggc agctcaggaa actgtattga agacatgtgt catggtattt ggttcggttg ggtgttgtgg gcagacgtcg gtggttgttt tcactagacg gtggaagatt ctctgcactg ttgttgctat ggccaagcat tgattcatgt tcgacaggct tattgcacgt aatcgtttgg gaggaatttc aataagcacc aatatgcgtg cttgcggtgg gtgttgtggc cagacgtcgt tggttgtttc cactagacgt tggaagat tc cggttggcgt catcatatat tgaaccacaa tttcatctcc tcaggctaca tagatgattc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 ttacattcat gataacgctg acaatcatac ggatcaggaa aaactcctgg gttatagaac cttccgagaa acatgggctt ggatctgcag aacatctacc ctgcaaactt caccagaact acatggactt gtagacacgg tgttgaattc cctctaccac aaaaagctgt gacgctgcag ttgagacaaa aaaaaccatc tcagagcagt tgttagtctg ccgattatca atcgattgca aggctacgaa ttggccacaa ttataactga cttgatcat <210> 16 <211> 387 <212> DNA <213> Ctenocephalides felis <220> <221> CDS <222> (20)..(262) <400> 16 gttaatttaa aataacaaa atg aaa gga aca tta tta ata tta tca tgt ctt 52 13 Met Lys Gly Thr Leu Leu Ile Leu Ser Cys Leu gtg atc atg ata agt gcc gaa tat gct gac gta gat gtg tgc caa. gat 100 Val Ile Met Ile Ser Ala Glu Tyr Ala Asp Val Asp Vai Cys Gin Asp 20 00ttg gac gat gga act ttt ctt gct gat tca aac aat tgc caa aat ftc 148 00Leu Asp Asp Gly Thr Phe Leu Ala Asp Ser Asn Asn Cys Gin Asn Phe 35 ttc att tgt gat gga ggc cga gct tgg aaa atg tat tgt cca gga tca 196 Phe Ile Cys Asp Giy Gly Arg Ala Trp Lys Met Tyr Cys Pro Gly Ser 50 rlctt tta tgg aat gat cac gaa gga. aca tgt gat tac gca caa aat gta 244 CILeu Leu Trp Asn Asp His Giu Gly Thr Cys Asp Tyr Ala Gin Asn Val 65 70 gaa tgt tac caa cca gaa taaaacattt taatatctga cagcgatttt 292 Giu Cys Tyr Gin Pro Giu ctgaaactat atttcatact actgttataa taaatttatc ttcattgctc tcctcctata 352 aatttattcc gttttaataa aatcaatata aagac 387 <210> 17 <2ii> 81 <212> PRT <213> Ctenocephaiides felis <400> 17 Met Lys Gly Thr Leu Leu Ile Leu Ser Cys Leu Val Ile Met Ile Ser 1 5 10 Ala Glu Tyr Ala Asp Val Asp Val Cys Gin Asp Leu Asp Asp Gly Thr 25 Phe Leu Ala Asp Ser Asn Asn Cys Gin Asn Phe Phe Ile Cys Asp Gly 40 Gly Arg Ala Trp Lys Met Tyr Cys Pro Gly Ser Leu Leu Trp Asn Asp 55 His Giu Gly Thr Cys Asp Tyr Ala Gin Asn Val Giu Cys Tyr Gin Pro 70 75 Giu <210> 18 <211> 387 <212> DNA <213> Ctenocephaiides felis <400> 18 gtctttatat tgattttatt aaaacggaat aaatttatag gaggagagca atgaagataa atttattata ttttattctg ttccataaaa aaattttggc tctacgtcag cctttcattt acagtagtat gttggtaaca gtgatcctgg aattgtttga catattcggc tgttatttta gaaatatagt ttctacattt acaatacatt atcagcaaga acttatcatg aattaac ttcagaaaat tgtacgtaat ttccaagctc aaagttccat atcacaagac cgctgtcaga tattaaaatg cacatgttcc ttcgtgatca ggcctccatc acaaatgaag cgtccaaatc ttggcacaca atgataatat taataatgtt 120 180 240 3'00 360 387 <210> 19 <211> 243 <212> DNA <213> Ctenocephalides felis <400> 19 atgaaaggaa. cattattaat gacgtagatg tgtgccaaga. caaaatttct tcatttgtga ttatggaatg atcacgaagg gaa attatcatgt cttgtgatca tgataagtgc Cgaatatgct tttggacgat ggaacttttc ttgctgattc aaacaattgc tggaggccga gcttggaaaa tgtattgtcc aggatcactt. aacatgtgat tacgcacaaa atgtagaatg ttaccaacca. 120 180 240 243 <210> <211> 243 <212> DNA <213> Ctenocephalides felis <400> ttctggttgg taacattcta taaaagtgat cctggacaat ttggcaattg tttgaatcag gtcagcatat tcggcactta cat <210> 21 <211> 960 <212> DNA <213> Ctenocephalides cattttgtgc gtaatcacat gttccttcgt gdtcattcca acattttcca agctcggcct ccatcacaaa tgaagaaatt 120 caagaaaagt tccatcgtcc aaatcttggc acacatctac 180 tcatg'atcac aagacatgat aatattaata atgttccttt 240 243 felis <400> 21 ttaatgtcga ccgcaccagg gtaaaaaagt cggaaatagg aaagtggata. aatgtccgtc gtgacgaaaa tacgtcataa cacagcctaa gaagaggaac gtgtcgggca agttcttcac cagtatttaa tatattaata. ttactttatg cggaacgcct agtagtacct aaaatttagt agcagatccg tctacagtgc accatcgaga. tagtatagga ttgcggacct ggtgcttcCg cgtattgcaa atgccgagca gtgtttacgt ccaagctact tattgatgaa. tgttatataa ttaacagtaa actcctgaac tgcgtgaatt gttaaggaat accactaaag ttttatccaa acccaatatt atggctctgg tacattggtg tgcgaagaac cgtggaaaat cctaaaggag caaagctgca gtattcaaca aaaaatatta tttagatttt ataaagaagt ctacacctgt cgtgcagttc cctgtagccc aaagtacagt atataaatga attgccccgc gcacaaaatg ataaatcatt ccgccgagtt ttgctttcaa agccagttcc acacaggaac aaagaaacta tggttgaaca atttgcatca accgaaaccc acccgaaatg agatcaagtg ctgcaaagta, ttgtcaaagc aaattttgca. ctatacagtt gtacgtcgta ttccccaagg gaacgacgca. tgatcaatgt ttgcgagagg tacaaaatat. caggctcgca ttggatgagc gagcaaaaat cagtattgtc tgcgctgatc caaggt ttca tattattatt tatgatccgt acatgtcctg tgtatggccg agcgaaacc t acatgccgga. ccgattggaa aaacctaaat gtactttgtt aa tat ga taa tgttctaatc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 ggcatttaag aattttacaa tttaaatcca tgaatatatt <210> 22 <211> 960 <212> DNA <213> Ctenocephalides felis 00 <400> 22 gattaga aca ttatcatatt aacaaagtac atttaggttt ttccaatcgg tccggcatgt aggtttcgct cggccataca caggacatgt acggatcata aataataata tgaaaccttg gatcagcgca gacaatactg atttttgctc gctcatccaa aatatattca tgcgagcctg atattttgta cctctcgcaa acattgatca tgcgtcgttc ccttggggaa tacgacgtac aactgtatag tgcaaaattt gctttgacaa tactttgcag cacttgatct catttcgggt gggtttcggt tgatgcaaat tggatttaaa aaaatctaaa ttgtaaaatt tgttcaacca tagtttcttt gttcctgtgt ggaactggct ttgaaagcaa aactcggcgg aa tgat ttat cattttgtgc gcggggcaat tcatttatat actgtacttt gggctacagg gaactgcacg acaggtgtag acttctttat taatattttt tgttgaatac tgcagctttg ctcctttagg attttccacg gttcttcgca caccaatgta ccagagccat aatattgggt ttggataaaa ctttagtggt attccttaac aattcacgca gttcaggagt ttactgttaa ttatataaca ttcatcaata agtagcttgg acgtaaacac tgctcggcat ttgcaatacg cggaagcacc aggtccgcaa tcctatacta tctcgatggt gcactg'taga cggatctgct actaaatttt aggtactact aggcgttccg cttaaatgcc cataaagtaa 120 tattaatata 180 ttaaatactg 240 gtgaagaact 300 tgcccgacac 360 gttcctcttc 420 ttaggctgtg 480 ttatgacgta 540 ttttcgtcac 600 gacggacatt 660 tatccacttt 720 cctatttccg 780 acttttttac 840 cctggtgcgg 900 tcgacattaa 960 <210> 23 <211> 1029 <212> DNA <213> Ctenocephalides Eel is <400> 23 tgttttatat atggtgagtt tggatgagcc agcaaaaatg agtattgtcc gcgc tgatca aaggtttcaa attattattg atgatccgtt catgtcctgc gtatggccgg gcgaaacctg catgccggaa cgattggaac aacctaaatt tactttgttt atatgataag 1020 gttctaatc 1029 cacattggtt taatgtcgac cgcaccagga taaaaaagta ggaaatagga aagtggatat atgtccgtca tgacgaaaat acgtcataat acagcctaag aagaggaacc tgtcgggcaa gttcttcacg agtatttaac atattaatat tactttatgt gcatttaaga tttattagtt ggaacgcctt gtagtaccta aaatttagtt gcagatccgg ctacagtgca ccatcgagat agtataggaa tgcggaccta g'tgcttccgt gtattgcaat tgccgagcac tgtttacgtc caagctactc attgatgaag gt tatataaa attttacaat ttgtggcgtt taacagtaaa ctcctgaacc gcgtgaattc t taaggaatc ccactaaaga tttatccaaa cccaatatta tggctctggg acat tggtga gcgaagaacc gtggaaaatt c taaaggaga aaagctgcaa tattcaacaa aaaatattat ttagattttt atctgtcgtt taaagaagta tacacctgta gtgcagttca ctgtagccca aagtacagtc tataaatgat ttgccccgca cacaaaatgc taaatcattg cgccgagttt tgctttcaag gccagttcct cacagg'aac t aagaaactat ggttgaacac ttaaatccat accgcttatg tttgcatcat ccgaaacccg cccgaaatgc gatcaagtgt tgcaaagtac tgtcaaagct aattttgcat tatacagtta tacgtcgtat t ccc caagga aacgacgcaa gatcaatgtc tgcgagagga acaaaatatg aggctcgcaa gaatatattt 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 <210> 24 <211> 1029 <212> DNA <213> Ctenocephalides felis <400> 24 gattagaaca ttatcatatt aacaaagtac atttaggttt ttccaatcgg tccggcatgt aatatattca tgcgagcctg atattttgta cctctcgcaa ac at tgat ca tgcgtcgttc tggatttaaa tgttcaacca tagtttcttt gttcctgtgt ggaactggct ttgaaagcaa aaaatctaaa taatattttt tgttgaatac tgcagctttg ctcctttagg attttccacg ttgtaaaatt ttatataaca ttcatcaata agtagcttgg acgtaaacac tgctcggcat ct taaa tgc c cataaagtaa tattaatata ttaaatactg gtgaagaac t tgcccgacac 120 180 240 300 360 00 aggtttcgct cggecataca caggacatgt acggatcata aataataata tgaaaccttg gatcagcgca gacaatactg atttttgcte gctcatccaa ac tcaccatc 1020 tataaaaca 1029 ccttggggaa tacgacgtac aactgtatag tgcaaaattt gctttgacaa tactttgcag cacttgatct catttcgggt gggtttcggt tgatgcaaat a taagcggta aactcggcgg aatgatttat cattttgtgc gcggggcaat tcatttatat actgtacttt gggetacagg gaactgcacg aeaggtgtag acttctttat acgacagata gttcttcgca caccaatgta ccagagccat aatattgggt ttggataaaa ctttagtggt attccttaac aa ttcacgca gttcaggagt ttactgttaa acgccacaaa ttgcaatacg cggaagcacc aggtccgcaa tcctatacta. tctcgatggt gcactgtaga cggatctgct actaaatttt aggtactact aggcgttccg actaataaaa gttcctcttC ttaggctgtg ttatgacgta ttttcgtcac gacggacatt tatccacttt cctatttccg acttttttac cctggtgcgg tcgacattaa accaatgtga, <210> <211> 1048 <212> DNA <213> Ctenocephalides felis <220> <221> CDS <222> (19)..(873) <400> gtaacatatt tattaaga atg ttt tat atc aca. ttg gtt ttt att Met Phe Tyr Ile Thr Leu Val Phe Ile agt ttt Ser Phe gtg gcg tta Val. Ala Leu gga aeg ect Gly Thr Pro gtc gtt ace get Val Val Thr Ala gat ggt gag ttt Asp Gly Glu Phe tta. aca. gta. aat Leu Thr Val Asn gaa gta ttt gca Glu Val Phe Ala tea Ser aat gte gac Asn V~al Asp ttg gat gag Leu Asp Glu gta ceg aaa Val Pro Lys eec gca Pro Ala eea gga gta gta. Pro Gly Val Val act cct gaa. cet Thr Pro Glu Pro aea. ect Thx Pro ec Pro gag eaa. aaa tgt Glu Gin Lys Cys aaa Lys 65 aaa gta. aaa ttt Lys Val Lys Phe agt Ser tgc gtg aat teg Cys Val Asn Ser 195 243 291 agt tea ccc gaa Ser Ser Pro Glu cag tat tgt ceg Gin Tyr Cys Pro ata gga. gca. gat Ile Gly Ala Asp ceg gtt Pro Val aag gaa. tee Lys Glu Ser eta eag tgc Leu Gin Cys 110 tgt Cys age eca. gat caa Ser Pro Asp Gin gtg Val 100 tge get gat eaa Cys Ala Asp Gin agt gga. tat Ser Gly Tyr 105 caa. ggt tte Gin Gly Phe ace act aaa gaa agt aca gte tge aaa. Thr Thr Lys Glu Ser Thr Val Cys Lys 115 gta Val 120 aaa tgt Lys Cys 125 ccg tea cca. tcg Pro Ser Pro Ser aga Arg 130 ttt tat eca. aat ata. aat gat tgt caa. Phe Tyr Pro Asn Ile Asn Asp Cys Gin 135 agc Ser 140 tat tat tat tgt gac gaa. aat agt ata. Tyr Tyr Tyr Cys Asp Glu Asn Ser Ile acc caa tat tat Thr Gin Tyr Tyr tgC Cys 155 00 ccc gca aat ttt Pro Ala Asn Phe gca Al a 160 tat gat ccg tta Tyr Asp Pro Leu cgt Arg 165 cat aat tgc gga His Asn Cys Giy cct atg Pro Met 170 483 531 579 627 gct ctg ggc Ala Leu Gly gtg ctt ccg Val Leu Pro 190 aca Thr 175 aaa. tgc tat aca Lys Cys Tyr Thr gtt Vai 180 aca. tgt cct gca Thr Cys Pro Ala cag cct aag Gin Pro Lys 185 tgt atg gcc Cys Met Ala tac att. ggt gat Tyr Ile Gly Asp tca ttg tac gtc Ser Leu Tyr Val gta Val 200 gga. aga Gly Arg 205 gga acc gta ttg Gly Thr Val Leu caa Gin 210 tgc gaa gaa. ccc Cys Giu Glu Pro gcc Al a 215 gag ttt tcc cca. Glu Phe Ser Pro agg Arg 220 agc gaa. acc tgt Ser Glu Thr Cys aag aac gac gca Lys Asn Asp Ala 240 9g9 caa tgc cga. Gly Gin Cys Arg gca. Al a 230 cgt gga aaa ttt Arg Gly Lys Phe gct Ala 235 675 723 771 ttC Phe aca. tgc cgg aag Thr Cys Arg Lys ttC acg tgt tta Phe Thr Cys Leu cgt cct Arg Pro 250 aaa. gga gag Lys Gly Glu caa gct act Gln Ala Thr 270 cca Pro 255 gtt cct gat caa Val Pro Asp Gin tgt Cys 260 ccg att gga. aca. Pro Ile Gly Thr gta. ttt aac Val Phe Asn 265 aaa cct aaa Lys Pro Lys 819 867 caa agc tgc aac Gin Ser Cys Asn gga. act tgc gag Gly Thr Cys Glu agg Arg 280 t ta Leu tat taatatattg atgaagtatt caacaaaaga aactatacaa aatatgtact Tyr 285 ttgttttact ttatgtgtta tataaaaaaa tattatggtt gaacacaggc tcgcaaatat 983 gataaggcat ttaagaattt tacaatttag atttttttaa 1043 atccatgaat atatttgttc taatc 1048 <210> 26 <211> 285 <212> PRT <213> Ctenocephalides felis <400> 26 Met Phe Tyr Ile Thr Leu Val Phe Ile Ser Phe 1 5 10 Val Ala Leu Ser Val Val Thr Ala Tyr Asp Gly Glu Phe Asn Vai Asp Gly Thr Pro Leu Thr 25 00 Vai Asn Lys Glu Val Phe Ala Ser Leu Asp Giu Pro Ala Pro Giv Val Val Lys Gin Pro Lys Ser Asp 145 Tyr Cys Gly Leu Val 225 Thr Pro Cys Pro Lys Tyr Asp Giu Arg 130 Giu Asp Tyr Asp Gin 210 Gly Cys Asp Asn Thr Pro Val Lys Cys Pro Gin Val 100 Ser Thr 115 Phe Tyr Asn Ser Pro Leu Thr Val 180 Lys Ser 195 Cys Glu Gin Cys Arg Lys Gin Cys 260 Thr Giy 275 Giu Phe Giu Cys Val Pro Ile Arg 165 Thr Leu Giu Arg Phe 245 Pro Pro Ser 70 Ile Ala Cys Asn Gly 150 His Cys TPyr Pro Ala 230 Phe Ile Thr 55 Cys G ly Asp Lys Ile 135 Thr Asn Pro Val Ala 215 Arg Thr Gly 40 Pro Val Aia Gin Val1 120 Asn Gin Cys Ala Val 200 Giu Giy Cys Thr Arg. 280 Val Asn Asp Ser 105 Gin Asp Tyr Gly Gin 185 Cys Phe Lys Leu Val 265 Pro Ser Pro 90 Gly Gly Cys Tyr Pro 170 Pro Met Ser Phe Arg 250 Phe Lys Cys 75 Val Tyr Phe Gin Cys 155 Met Lys Ala Pro Ala 235 Pro Asn Pro Ser Lys Leu Lys Ser 140 Pro Ala Val Gly Arg 220 Phe Lys Gin Giu Ser Giu Gin Cys 125 Tyr Ala Leu Leu Arg 205 Ser Lys Gly Ala *Gin Pro Ser Cys 110 Pro Tyr Asn Gly Pro 190 Gly Giu Asn Glu Thr 270 Lys Cys Giu Met Cys Ser Thr Thr Ser Pro Tyr Cys Phe Ala 160 Thr Lys 175 Tyr Ile Thr Val Thr Cys Asp Ala 240 Pro Val 255 Gin Ser Thr Cys Giu Lays Pro Lys Leu <210> 27 <211> 1048 <212> DNA <213> Ctenocephalides felis <400> 27 gattagaaca aatatattca tggatttaaa aaaatctaaa ttgtaaaatt cttaaatgcc ttatcatatt tgcgagcctg tgttcaacca taatattttt ttatataaca cataaagtaa 120 aacaaagtac atattttgta tagtttcttt tgttgaatac ttcatcaata tattaatata 180 atttaggttt cctctcgcaa gttcctgtgt tgcagctttg agtagcttgg ttaaatactg 240 ttccaatcgg tccggcatgt aggtttcgct c ggc cat ac a caggacatgt acggatcata aataataata tgaaaccttg gatcagcgca gacaatactg at ttttgc tc gctcatccaa actcaccatc 1020 tataaaacat 1048 acattgatca tgcgtcgttc cct tggggaa tacgacgtac aactgtatag tgcaaaattt gctttgacaa tactttgcag cacttgatct catttcgggt gggtttcggt tgatgcaaat ataagcggta ggaactggct ttgaaagcaa aac tcggcgg aatgatttat cattttgtgc gcggggcaat tcatttatat actgtacttt gggcta cagg gaactgcacg acaggtgtag acttctttat acgacagata ctcctttagg attttccacg gttcttcgca caccaatgta ccagagccat aatattgggt ttggataaaa ctttagtggt attccttaac aattcacgca gttcaggagt ttactgttaa acgccacaaa acgtaaacac tgctcggcat ttgcaatacg cggaagcacc aggtccgcaa tcctatacta tctcgatggt gcactgtaga cggatctgct actaaatttt aggtactact aggcgttccg actaataaaa gtgaagaact tgcccgacac gttcctcttc ttaggctgtg ttatgacgta ttttcgtcac gacggacatt tatccacttt cctatttccg actttttac cctggtgcgg tcgacattaa accaatgtga 300 360 420 480 540 600 660 720 780 840 900 960 tcttaataaa tatgttac <210> 28 <211> 855 <212> DNA <213> Ctenocephalides felis <400> 28 atgttttata gatggtgagt ttggatgagc gagcaaaaat cagtattgtc tgcgctgatc caaggtttca tattattat tatgatccgt acatgtcctg tgtatggccg agcgaaacct acatgccgga ccgattggaa aaacctaaat tcacattggt ttaatgtcga CCgcaccagg gtaaaaaagt cggaaatagg aaagtggata aatgtccgtc gtgacgaaaa tacgtcataa cacagcctaa gaagaggaac gtgtcgggca agttcttcac cagtatttaa tatat ttttattagt cggaacgcct agtagtacct aaaatttagt agcagatccg tctacagtgc accatcgaga tagtatagga ttgcggacct ggtgcttccg cgtattgcaa atgccgagca gtgtttacgt ccaagctact tttgtggcgt ttaacagtaa actcctgaac tgcgtgaatt gttaaggaat accactaaag ttttatccaa acccaatatt atggctctgg tacattggtg tgcgaagaac cgtggaaaat cctaaaggag caaagc tgca tatctgtcgt ataaagaagt ctacacctgt cgtgcagttc cctgtagccc aaagtacagt atataaatga attgccccgc gcacaaaatg ataaatcatt ccgccgagtt ttgctttcaa agccagttcc acacaggaac taccgc itat atttgcatca accgaaaccc acccgaaatg agatcaagtg ctgcaaagta ttgtcaaagc aaattttgca ctatacagtt gtacgtcgta ttccccaagg gaacgacgca tgatcaatgt ttgcgagagg 120 180 240 300 360 420 480 540 600 660 720 780 840 855 <210> 29 <211> 855 <212> DNA <213> Ctenocephalides felis <400> 29 atataattta tactgttcca gaacttccgg gacacaggt tcttccggcc c tgtgcagga acgtaacgga gtcacaataa acatttgaaa actttgatca ttccggacaa tttacattt tgcgggctca ggtttcctct atcggacatt catgttgcgt tcgctccttg a taca tac ga catgtaactg tcatatgcaa taatagcttt ccttgtactt gcgcacactt tactgcattt tgctcgggtt tccaatgatg cgcaagttcc gatcaggaac cgttcttgaa gggaaaactc cgtacaatga tatagcattt aatttgcggg gacaatcatt tgcagactgt gatctgggct cgggtgaact tcggtacagg caaatacttc cggtaacgac tgtgttgcag tggctctcct agcaaatttt ggcgggttct tttatcacca tgtgcccaga gcaataatat tatatttgga actttcttta acaggattcc gcacgaattc tgtaggttca tttat ttac t agataacgcc cttgagtag ttaggacgta ccacgtgctc tcgcattgca atgtacggaa gccataggtc tgggttccta taaaatctcg gtggtgcact ttaaccggat acgcaactaa ggagtaggta gttaaaggcg acaaaactaa cttggttaaa aacacgtgaa ggcattgccc atacggttcc gcaccttagg cgcaattatg tactatitc atggtgacgg gtagatatcc ctgctcctat attttactt ctactcctgg ttccgtcgac taaaaaccaa 120 180 240 300 360 420 480 540 600 660 720 780 840 attaaactca ccatcataag tgtgatataa aacat 00 <210> <211> 802 <212> DNA <213> Ctenocephalides felis <220> <221> CDS <222> (799) <400> t tat gat ggt gag ttt aat gtc gac gga acg cot tta aca gta aat aaa 49 Tyr Asp Gly Glu Phe Asn Val Asp Gly Thr Pro Leu Thr Val Asn Lys gaa gta ttt Glu Val Phe cct gaa cct Pro Giu Pro gca Ala tca ttg gat Ser Leu Asp gag ccc Giu Pro 25 gca cca gga gta Ala Pro Gly Val gta cot act Val Pro Thr aaa aaa gta Lys Lys Val 97 145 aca cot gta Cog Thr Pro Val Pro ccc gag caa aaa Pro Glu Gin Lys aaa ttt Lys Phe agt tgc gtg aat Ser Cys Val Asn tog Ser 55 tgc agt tca coo Cys Ser Ser Pro gaa Glu atg cag tat tgt Met Gin Tyr Cys cog Pro gaa ata gga gca Gu Ile dly Ala cog gtt aag gaa Pro Val Lys Giu too Ser tgt ago oca gat Cys Ser Pro Asp gtg tgo got gat Val Cys Ala Asp caa Gin agt gga tat eta Ser Gly Tyr Leu cag Gin 90 tgo aco act aaa Cys Thr Thr Lys gaa agt Giu Ser aca gte tge Thr Val Cys tat oca. aat Tyr Pro Asn 115 aaa Lys 100 gta eaa ggt tto Val Gin Gly Phe aaa Lys 105 tgt cog tea oca Cys Pro Ser Pro tog aga ttt Ser Arg Phe 110 gao gaa aat Asp Glu Asn 337 385 ata aat gat tgt Ile Asn Asp Cys ago tat tat tat Ser Tyr Tyr Tyr agt ata. Ser Ile 130 gga ace oaa tat Gly Thr Gin Tyr tat Tyr 135 tgo coo gca aat Cys Pro Ala Asn ttt1 Phe 140 gca tat gat cog Ala Tyr Asp Pro t ta Leu 145 ogt cat aat tgc Arg His Asn Cys gga Gly 150 cct atg got ctg Pro Met Ala Leu aca aaa tgc tat Thr Lys Cys Tyr gtt aca. tgt cot Val Thr Cys Pro gca Ala 165 cag cct aag gtg Gin Pro Lys Val ott Leu 170 cog tao att ggt Pro Tyr Ile Gly gat aaa Asp Lys 175 tea ttg tao Ser Leu Tyr gte Val 180 gta tgt atg gee Val Cys Met Ala gga aga Gly Arg 185 gga ace gta Gly Thr Val ttg caa tgc Leu Gin Cys 190 00 gaa Glu tgC Cys aag Lys 225 tgt Cys gga Gly gaa. Giu cga. Arg 210 ttC Phe ccg Pro act Thr CCC Pro 195 gca Ala ttC Phe att Ile tgC Cys gcc gag ttt Ala Glu Phe Cgt Arg acg Thr gga Gly gag Glu 260 gga. Gly tgt Cys aca. Thr 245 agg Arg aaa Lys tta Leu 230 gta Val aaa Lys tcC Ser ttt Phe 215 cgt Arg ttt Phe cct Pro agg Arg ttC Phe aaa Lys caa Gin t ta Leu 265 agc gaa Ser Glu aag Lys gga Gly gc t Ala 250 tat Tyr aac Asn gag Giu 235 act Thr taa acc tgt Thr Cys 205 gac gca Asp Ala 220 cca gtt Pro Val caa agc Gin Ser gtc Val aca. Thr cct Pro tgC Cys ggg caa Gly Gin tgc cgg Cys Arg gat caa Asp Gin 240 aac ace Asn Thr 255 625 673 721 769 <210> 31 <211> 266 <212> PRT <213> Ctenocephaiides felis <400> 31 Tyr 1 Giu Pro Lys Pro Val Thr Tyr Ser Leu 145 Val Asp Val Glu Phe Glu Cys Vai Pro Ile 130 Arg Thr Giy Phe Pro Ser Ile Ala Cys Asn 115 Gly His Cys Giu Phe Asn Val Ala Thr Cys Gly Asp Lys 100 Ile Thr Asn Pro 5 Ser Pro Val Ala Gin Val Asn Gin Cys Al a 165 Leu Val Asn Asp 70 Ser Gin Asp Tyr Gly 150 Gin Asp Pro Ser 55 Pro Gly Gly Cys Tyr 135 Pro Pro Asp Glu Lys Cys Val Tyr Phe Gin 120 Cys Met Lys Giy Thr Pro Leu Thr Val Asn Lys 10 Pro 25 Pro Ser Lys Leu Lys 105 Ser Pro Ala Val Al a Glu Ser Glu Gin 90 Cys Tyr Ala Leu Leu Pro Gin Pro Ser 75 Cys Pro Tyr Asn Gly 155 Pro Gly Lys Giu Cys Thr Ser Tyr Phe 140 Thr Tyr Val Lys Gin Pro Lys Ser 110 Asp Tyr Cys Gly Pro Lys Tyr Asp Glu Arg Giu Asp Tyr Asp 175 Thr Val Cys Gin Ser Phe Asn Pro Thr 160 Lys 170 Ser Leu Tyr Val Val Cys Met Ala Gly Arg Gly Thr Val Leu Gin Cys Glu, Glu Pro 195 Cys Arg Ala 210 Ala Glu Phe Ser Arg Ser Glu Thr Val Gly Gin Arg Gly Lys Phe 215 Ala Phe Lys Asn Asp 220 Ala Thr Cys Arg Lys Phe Phe Thr Cys Leu 225 230 Arg Pro Lys Gly Glu 235 Pro Val Pro Asp Gin 240 Cys Pro Ile Gly Thr Cys Gly Thr 245 Glu Arg 260 Val. Phe Asn Gin Ala 250 Thr Gin Ser Cys Asn Thr 255 Lys Pro Lys Leu Tyr 265 <210> 32 <211> 802 <212> DNA <213> Ctenocephalides felis <400> 32 ttaatataat aaatactgtt gaagaacttc cccgacacag tcctcttccg aggctgtgca atgacgtaac ttcgtcacaa. cggacatttg tccactttga tatttccgga ttttttacat tggtgcgggc gacattaaac ttaggtttcc ccaatcggac cggcatgttg gtttcgctcC gccatacata ggacatgtaa ggatcatatg taataatagc aaaccttgta tcagcgcaca caatactgca ttttgctcgg tcatccaatg tcaccatcat tctcgcaagt attgatcagg cgtcgttctt ttggggaaaa cgacgtacaa ctgtatagca caaaatttgc tttgacaatc ctttgcagac cttgatctgg tttcgggtga gtttcggtac atgcaaatac aa tcctgtgttg aactggctct gaaagcaaat ctcggcgggt tgatttatca ttttgtgccc ggggcaataa atttatatt t tgtactttct. gctacaggat actgcacgaa aggtgtaggt t tc t ttat t t cagctttgag cctttaggac tttccacgtg tcttcgcatt. ccaatgtacg a gagc c atag tattgggttc ggataaaatc ttagtggtgc tccttaaccg ttzcacgcaac tcaggagtag actgttaaag tagcttggtt gtaaacacgt ctcggcattg gcaatacggt gaagcacctt gtccgcaatt ctatactatt tcgatggtga actgtagata gatctgctcc taaattttac gtactactcc gcgttccgtC 120 180 240 300 360 420 480 540 600 660 720 780 802 <210> 33 <211> 436 <212> DNA <213> Ctenocephalides felis <220> <223> At location 347, n unknown <400> 33 cttgtatata tacacctctg gccgcctctt caaaggatac ttgtcctggt, ggagtgccta tgcgtgcaga gtgtgagact agtgagtttt cctcctccaa ttttcatgcg tacaaatgtg tctacttact. agaaatcc tc gcggtcattt. ctctgg accaaagatg ttgttccgac tgcaggaagg ctttgaaagc tcagcagcac cagcgccttg gcagatccag tgtgggccct tactacgact. gatgtttata aggcggagga gtatcagcaa gcaaccacct cagacaaaac acatattcgg acgacaacta gatccctatg ttcagtgtag tgtgtggtcg acgccgncgc atgcaaagta aatgtttaat caccagcacc acagcacttg cgcgttacaa cttccctttc catatacaac tattttgaat <210> 34 00 <211> 485 <212> DNA <213> Ctenocephalides felis <400> 34 ctcatttgca tggaggaaat ttaccaacaa accgccacca ggattacact attcgacgtg gtgcgtacct catccttacc ttggc gatccagcag ttcaacgtgg tgcgtgcaag ggaccaggac gataattctt gcccgatatg gcataccagt atcttaccat acaaaacatg gtcgttttac cgcctttatc catcgcaacc gcaaatggta c ttgtgcagc cagactgttt ctttccaacc caaagtatat atgcgccgga cgagtgcttg ctttgcttgc ctacgagtgc tgagttatat aggtgcaagt ttgcaacatc tttgaatgtg cagacatact gcgacagctg gtgcggacag acattaaacg ttcaacagcg gtcacttctt ttcccaacag tgagactctc tcagcgcgct cacctccacc gattgttttt ctcaaggtgt ttctgcagca cccatcatac caggattctt 120 180 240 300 360 420 480 485 <210> <211> 550 <212> DNA <213> Ctenocephalides felis <400> gtcgttttac cgcctttatc catcgcaacc gcaaatggta cttgtgcagc cagactgttt catctttccc ctcttgatat attaaattct tataaggtat atgcgccgga cgagtgc ttg ctttgcttgc ctacgagtgc tgagttatat aggtgcaagt aaccttgcca gcaaacaaaa acatgtccta cagacatact gcgacagc tg gtgcggacag acattaaacg ttcaacagcg gtcacttctt acatcttccc actggaacta gtgagtgtgt tcagcgcgct cacctccacc gattgttttt ctcaaggtgt ttctgcagca ccccatccat caacagcagg aatgtacaaa atatgaaatg ttaccaacaa ac cgc Cac ca ggattacact attcgacgtg gtgcgtacc t accatcctta atttcctttt aggagaagtt ctattgatat tgcgtgcaag ggaccaggac gataattctt gcccgatatg gcataccagt ccatccttac ggccgaaaat tctaaagacg acatcaagct <210> 36 <211> 436 <212> DNA <213> Ctenocephalides felis <400> 36 taccatcttt aatctcttga acgatt taaa catacaagct atggttttaa ataagaggcc atgtagtagt tttatcacta cccaaccttg tatgcaaaca ttctacatgt taataaaggt atttattttt tgatatccat tggtatagac ttattt ccaacatctt aaaactggaa acc tagtgag tatggtgatt tatttaatat aatattattg aatttactta ccccaacagc ctaaatgtac tgtgttatat tatttcattt taaatagatt aatattaaga ataattatga aggatttcct aaaaggagaa gaaattgcta aaaggcaatg aattaaaaat agtgacggat gtggcatcat tttgaccgaa gtttctaaag ttgcatatta tatcggttta ctataaagtg agatctgata tatgcaatga <210> 37 <211> 498 <212> DNA <213> Ctenocephalides felis <220> <223> At locations 23, 30, 33, 55, 130, 138, 147, 154, 158, 383 and 457, n unknown <400> 37 aaaaggagaa gtttctaaag acnatttttn ttntacatgt acctagtgag tgtgnggggg 00 ggaaattgct taaaggcaan taattaaaaa aagtgacgga agttgcatca tgcaactttt tatgaaaata tattaagtta attgcatatt gtatcggntt tctataaagt tagatctgat ttatgcaatg aataaaacgc aaaataaaga agaaa t tt acatacaagc aatggcnnta gataagaggc aatgtagtag Atttatcact atnttttatt actttataca ttaataaagg aatntatntt ctgatatcca ttgttataga attatttatt gttttaagta aaagctnttt ttatggtgat ttatttaata taatattatt caatttactt caacatttta taaatcttat tatcaatatg ttatttcatt ttaaatagat gaatattaag aataattatg tttaactgct tagggcacaa. CttcttgCgc <210> 38 <211> 436 <212> DNA <213> Ctenocephalides fells <220> <223> At locations 64, 86, 175, 360, 385, 390 and 429, nl unknown <400> 38 aataaaattt aggncttatt atgcgtttta atcattgcat ttatcagatc tcactttata tttaaccgat aatatgcant cttaacttaa tttattttca ttaaaagttg aatgatgcaa tatccgtcac gatttttaat ccattgcctt agcaat tagcgcaaga tattgngccc caagcagtta ctcataatta ttcttaatat taatctattt taaangaaan agcatattgt taataagatt aataaaatgt ttaagtaaat tcaataatat aatattaaat aaataccata ataaaaaaag tatacttaaa tgaataaata tggctataac tatggatatc aaaaaataaa acctttatta cttttgttaa acaataaaaa atagngataa aactactaca, aggcctctta ttttaaaccn agcttgttgt 120 180 240 300 360 420 436 <210> 39 <211> 1513 <212> DNA <213> Ctenocephaiides felis <220> <221> CDS <222> (1)..(1017) <220> <223> At locations 1356, 1430, 1491 unknown and 1495, n <400> 39 tac aag tgc Tyr Lys Cys 1 tta tgc cca Leu Cys Pro ggc cct aca Gly Pro Thr gtt ccg act Val Pro Thr aaa ctt ggt cta aat ggt Lys Leu Gly Leu Asn Gly 5 ctg caa agc ggt Leu Gin Ser Gly cat ttt His Phe tgg Trp aac ttg tat ata Asn Leu Tyr Ile gag ttt tac caa Glu Phe Tyr Gin aga tgt gtg Arg Cys Val cct cca att Pro Pro Ile tat tcg gaa tgt Tyr Ser Glu Cys tta Leu att aca cct ctg Ile Thr Pro Leu 96 144 192 act acg act Thr Thr Thr aca act aca cca Thr Thr Thr Pro gca Ala ccg ccg cct ctt Pro Pro Pro Leu ttt tca tgc gtg cag gaa ggg atg ttt ata. gat ccc tat gac agc act24 240 00 Phe tgC Cys gta Val cag Gln gog Ala agc Ser 145 gaa Glu tgc Cys gcg Ala cca Pro ttt Phe 225 tta Leu gag Glu tca S er tta Leu Ser Cys Val Gin Glu Gly Met Phe Ie Asp Pro Tyr Asp Ser Thr aaa Lys gog Ala o aa Gin cct Pro 130 ggo Gly tgt Cys gcc Al a cct Pro gga Gly 210 ttg Leu aac Asn tta Leu gac Asp cca Pro 290 gga Gly cgt Arg tgt Cys 115 t gg Trp tca Ser gtg Val gga Gly tta Leu 195 cca Pro gat Asp got Ala tat Tyr tgt Cys 275 tcc Ser tao Tyr tac Tyr 100 gtg Val 0 aa Gin ttt Phe aga Arg cag Gin 180 tcc Ser gga Gly tac Tyr o aa Gin t tc Phe 260 tta Leu tta Leu tac Tyr aat Asn gtc Val cca. Pro gca Ala ctc Leu 165 aca Thr gag Glu cca Pro act Thr ggt Gly 245 aac A.sn ggt cca Pro aaa Lys tgt Cys gc t Ala cct Pro gat Asp 150 tot Ser tac Tyr tgc Cys tog Ser gat Asp 230 gta Val agc Ser gca Ala tot Ser tgt Cys cot Pro tcc Ser acg Thr 135 cca Pro gga Gly ttc Phe ttg Leu caa Gin 215 aat Asn ttc Phe gt t Val agt Ser ttc Phe 295 got Ala ggt Gly ctt Leu 120 ccg Pro gca Ala gga Gly agc Ser gcg Ala 200 ccc Pro tct Ser gac Asp ctg Leu gtc Val 280 cca Pro ttg Leu tct Ser 105 tog Ser cog Pro gao Asp aat Asn gcg Ala 185 aca Thr ttt Phe tgo Cys gtg Val cag Gin 265 act Thr aoo Thr aaa Lys 90 act Thr gag Giu coo Pro aaa Lys ttc Phe 170 ott Leu got Ala got Ala aaa Lys gocc Al a 250 cag Gin tot. Ser ttg Leu ga Al a tac Tyr tgo Cys ata Ile aca Thr 155 aac Asn tao Tyr gca Ala tgo Cys tgg Trp 235 oga Arg tgo Cys too Ser cca Pro ggc C ly t to Phe ota Leu tao Tyr 140 tgo Cys gtg Val caa Gin ct Pro gtg Val 220 tao Tyr tat Tyr gta Val cca Pro aca Thr 300 gga Gly ago Ser aga Arg 125 dac Asn aaa Lys ggt C ly caa Gin cca Pro 205 ogg Arg tao Tyr got. Ala cot Pro too Ser 285 tot. Ser gga Gly ago Ser 110 aat Asn tgc Cys gta Val cgt Arg t go Cys 190 oca Pro aca Thx gag Glu tgt Cys gca Ala 270 ata Ile too Ser ttc Phe aog Thr cot Pro gtg Val1 tat ttt Phe 175 gtg Val cg Pro gga Gly tgo Cys goa Ala 255 tac Tyr oca Pro coa Pro agt Ser tat Tyr ooa Pro cag Gin ttt Phe 160 aoa Thr caa Gin oca Pro ttg Leu aoa Thr 240 got Ala cag Gin too Ser aca Thr 288 336 384 432 480 528 576 624 672 7120 768 816 864 912 960 goa gga ttt cot ttt ggc oga aaa tot ott. gat atg oaa aca aaa act Ala Gly Phe Pro Phe Gly Arg Lys Ser Leu Asp Met Gln Thr Lys Thr 305 310 315 320 gga act aaa tgt aca aaa gga gaa gtt tct aaa gac gat tta aat tct 1008 Gly Thr Lys Cys Thr Lys Gly Glu Val Ser Lys Asp Asp Leu Asn Ser 325 330 335 00 aca. tgt acc tagtgagtgt gttatatgaa attgctattg catattacat 1057 Thr Cys Thr acaagcttaa taaaggttat ggtgatttat ttcatttaaa ggcaatgtat cggtttaatg 1117 gttttaaatt tattttttat ttaatattaa atagattaat taaaaatcta taaagtgata CK1 1177 agaggcctga tatccataat attattgaat attaagaagt gacggataga tctgataatg 1237 tagtagttgg tatagacaat ttacttaata attatgagtg gcatcattat gcaatgattt 1297 atcactatta tttattcaac attttattta actgcttgca acttttaata aaacgcatnt 1357 tttattgttt taagtataaa tcttattagg gcacaatatg aaaataaaaa taaagaactt 1417 tatacaaaag ctnttttatc aatatgcttc ttgcgctatt aagttaagaa attttattta 1477 tgctgggaaa aaancaanaa atgctttatt tattct 1513 <210> <211> 339 <212> PRT <213> Ctenocephaiides felis <223> At locations 1356, 1430, 1491 and 1495, n unknown <400> Tyr Lys Cys Lys Leu Gly Leu Asn Gly Ala Leu Gin Ser Gly His Phe 1 5 10 Leu Cys Pro Trp Asn Leu Tyr Ile Ser Glu Phe Tyr Gin Arg Cys Val 25 Gly Pro Thr Tyr Ser Glu Cys Leu Ile Thr Pro Leu Pro Pro Pro Ile 40 Vai Pro Thr Thr Thr Thr Thr Thr Thr Thr Pro Ala Pro Pro Pro Leu 55 Phe Ser Cys Val Gin Giu Gly Met Phe Ile Asp Pro Tyr Asp Ser Thr 70 75 Cys Lys Gly Tyr Tyr Lys Cys Ala Leu Lys Ala Gly Gly Gly Phe Ser 90 Val Ala Arg Tyr Asn Cys Pro Gly Ser Thr Tyr Phe Ser Ser Thr 100 105 110 Tyr 00 Gin Ala Ser 145 Glu Cys Ala Pro Phe 225 Leu Glu Ser Leu Ala 305 Gly Thr Gin Pro 130 Gly Cys Ala Pro Gly 210 Leu Asn Leu Asp Pro 290 Gly Thr Cys Cys Val Val 115 Trp Gin Pro Ser Phe Ala Val Arg Leu 165 Gly Gin Thr 180 Leu Ser Glu 195 Pro Gly Pro Asp Tyr Thr Ala Gin Gly 245 Tyr Phe Asn 260 Cys Leu Gly 275 Ser Leu Pro Phe Pro Phe Lys Cys Thr 325 Thr Ala Pro Asp 150 Ser Tyr Cys Ser Asp 230 Val Ser Al a Ser Gly 310 Ser Thr 135 Pro Gly Phe Leu Gin 215 Asn Phe Val Ser Phe 295 Arg Leu 120 Pro Ala Gly Ser Ala 200 Pro Ser Asp Leu Val 280 Pro Lys Ser Pro Asp Asn Ala 185 Thr Phe Cys Val Gin 265 Thr Thr Ser Giu Pro Lys Phe 170 Leu Ala Ala Lys Al a 250 Gin Ser Leu Leu Ser 330 Cys Leu Arg Asn Pro Pro Ile Thr 155 Asn Tyr Ala Cys Trp 235 Arg Cys Ser Pro Asp 315 Tyr 140 Cys Val Gin Pro Val 220 Tyr Tyr Val1 Pro Thr 300 Met 125 Asn Lys Gly Gin Pro 205 Arg Tyr Ala Pro Ser 285 Ser Gin Cys Val Arg Cys 190 Pro Thr Glu Cys Ala 270 Ile Ser Thr Val Gin Tyr Phe 160 Phe Thr 175 Val Gin Pro Pro Gly Leu Cys Thr 240 Ala Ala 255 Tyr Gin Pro Ser Pro Thr Lys Thr 320 Lys Gly Giu Val Lys Asp Asp Leu Asn Ser 335 <210> 41 <211> i513 <212> DNA <213> Ctenocephaiides fells <220> <223> At locations 19, 23, 84 and 158, n unknown <400> 41 agaataaata aagcatttnt tgnttttttc ccagcataaa taaaatttct taacttaata 00 gcgcaagaag ttgtgcccta agcagttaaa cataattatt cttaatattc atctatttaa aatgaaataa tataacacac tacatttagt ctgttgggga atggggaaga gcagaacgct cttgagcgtt acaatcctgt gaggtgcagc cgctgaagta 1020 gtctcacaca. 1080 tctgcacgca 1140 ttaggcactc 1200 aaccaggaca. 1260 agtatccttt 1320 aaagaggcgg 1380 gcagaggtgt 1440 ttatatacaa 1500 gtttgcactt 1513 cat atz tap aac aa t tat atc tca tcC aga agt gt t taa ccg tgt tgt :attgata Lagatt ta Laatgttg rtaaattg :aatatta :taaataa :accataa ctaggta :agttttt .tgttggc .gacactt .gaaatat .tgtgcac cacgcaa cgccaag ctgtccg aaanagcttt tac ttaaaac aataaataat tctataccaa tggatatcag aaaataaatt cctttattaa catgtagaat gtttgcatat aaggttggga gcacctaaac aactcagctg tcgtagtacc gcaaagggt t cactcggata gcgcatgtaa tgtataaagt aataaaanat agtgataaat ctactacatt gcctcttatc taaaaccatt gc ttgtatgt t taaatcgtc caagagattt aagatggtaa agtctgactg cacaagcata atttgcaaga gcgatggtcc aaggcgcttg aacgacccac tctttatttt gcgttttatt cattgcataa. atcagatcta actttataga aaaccgatac aatatgcaat tttagaaact tcggccaaaa. ggatggta ag gtatgcaggt tcgggccacg attatcagtg tggtcctggt cacgcattgt gttgaaattt tattttcata aaaagttgca tgatgccact tccgtcactt tttttaatta attgccttta agcaatttca tctccttttg ggaaatcctg gatggtatgg acgcactgct tcgaatacac taatccaaaa ggcggtggtg tggtaaagcg cctccagaga 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 ttcaaaatat actttgcatg ttttgtctgc tggatctgca aatgagccgc gttgtatatg ggcggcggcg taggtggttg ccaaggcgct ggaggatttc cgaaagggaa gcgaccacac attgctgata cgtgctgctg aagtaagtag attgtaacgc gctacactga atcctccgcc tgctttcaaa gcacatttgt gcaagtgctg tcatagggat ctataaacat cccttcctgc acgcatgaaa cggtgctggt: gtagttgtcg tagtcgtagt agtcggaaca attggaggag aattaaacat tccgaatatg tagggcccac acatctttgg taaaactcac gttccatggg cataaaaaat gaccgctttg cagagcacca tttagaccaa gta <210> <211> <212> <213> <220> <221> <222> <220> <223> 42 1832 DNA Ctenocephalides felis CDS (146)..(1336) At locations 3, unknown 1675, 1749, 1810 and 1814, n ggcaagcagt; gggtaacaac gcagagtacg cgggggattt <400> 42 tcnaatacga ctcactatag agtaacttca aatttttggt acaatatcat tatcaaaaaa attgtttatt ttaaagtgga 120 atCtgttaaa tcgcgtttca ttacg atg aaa ctg ctt tta gtg ctc ttg gct Met Lys Leu Leu Leu Val Leu Leu Ala 1 acc ttc tcg tgt aca. ttg tct ttg gag tct caa gaa cta att cca gtt 220 Thr Phe Ser Cys Thr Leu Ser Leu Glu Ser Gin Glu Leu Ile Pro Val gaa aca aat tcc agc agc gtt cgt att Giu Thr Asn Ser Ser Ser Val Arg le cag caa cca ttc Gin Gin Pro Phe att tgt Ile Cys cca. aga gtt Pro Arg Vai tac tac aag Tyr Tyr Lys gga Gly cta ttt cgc gat Leu Phe Arg Asp ttg gat cc t aca Leu Asp Pro Thr tgc aaa agg Cys Lys Arg agc ggt cat Ser Giy His 316 364 tgc aaa. ctt ggt Cys Lys Leu Giy aat ggt gct ctg Asn Gly Ala Leu c aa Gin ttt tta Phe Leu tgc cca tgg aac Cys Pro Trp Asn tat ata agt gag Tyr Ile Ser Giu ttt tac caa aga tgt Phe Tyr Gin Arg Cys cct ctg cct ccL cca. Pro Leu Pro Pro Pro ggc cct aca tat Giy Pro Thr Tyr gaa tgt tta att Glu Cys Leu Ile ac a Thr 100 att gtt ccg act Ile Val Pro Thr act Thr 110 acg act acg aca Thr Thr Thr Thr act Thr 115 aca cca gca ccg Thr Pro Ala Pro ccg cct Pro Pro 12 0 ctt ttt tca Leu Phe Ser act tgc aaa Thr Cys Lys 140 tgc Cys 125 gtg cag gaa. ggg Val Gin Giu Giy ttt ata gat ccc Phe Ile Asp Pro tat gac agc Tyr Asp Ser 135 gga gga ttc Gly Gly Phe 556 604 gga tac tac aaa Gly Tyr Tyr Lys tgt Cys 145 gct ttg aaa gca Aia Leu Lys Aia ggc Gly 150 agt gta. Ser Vai 155 gcg cgt tac aat Ala Arg Tyr Asn cct ggt tct act Pro Gly Ser Thr ttc agc agc acg Phe Ser Ser Thr tat Tyr 170 cag caa tgt gtg Gin Gin Cys Val gct tcc ctt tcg Ala Ser Leu Ser gag Giu 180 tgc cta aga aat Cys Leu Arg Asn 652 700 748 cca gcg cct tgg Pro Ala Pro Trp, c aa Gin 190 cca cct acg ccg Pro Pro Thr Pro ccc ata tac aac Pro Ile Tyr Asn tgc gtg Cys Val 200 cag agc ggc Gin Ser Gly ttt gaa tgt Phe Glu Cys 220 tca Ser 205 ttt gca gat cca Phe Ala Asp Pro gca Ala 210 gac aaa aca tgc Asp Lys Thr Cys aaa gta tat Lys Val Tyr 215 ggt cgt ttt Gly Arg Phe gtg aga. ctc Lct Val Arg Leu Ser gga aat ttc aac Gly Asn Phe Asn aca tgc Thr Cys 235 gcc gga cag aca Ala Gly Gin Thr tac Tyr 240 ttc agc gcg ctt Phe Ser Ala Leu tac Tyr 245 caa caa tgc gtg Gin Gin Cys Val 892 940 caa Gin 250 gcg cct tta tcc Ala Pro Leu Ser gag Giu 255 tgc ttg gcg aca Cys Leu Ala Thr gc t Ala 260 gca cct cca cca Ala Pro Pro Pro ccg Pro 265 oca cca gga cca Pro Pro Gly Pro 00 ttg ttt 1036 Leu Phe aca tta 1084 Thr Leu gct gag 1132 Ala Giu 315 cag tca 1180 Gin Ser 330 tcc tta 1228 Ser Leu aca gca 1276 Thr Ala act gga 1324 Thr Gly tct aca 1376 Ser Thr

395- ttg Leu aac Asn 300 tta Leu gac Asp cca Pro gga. Gly act Thr 380 tgt Cys gat Asp 285 gc t Ala tat Tyr tgt Cys tcc Ser ttt Phe 365 aaa Lys acc Thr gga cca tcg caa ccc ttt gct tgc gtg cgg aca gga 988 Gly Pro Ser Gin Pro Phe Ala Cys Val Arg Thr Gly 270 275 280 tac act gat aat tct tgc aaa tgg tac tac gag tgc Tyr Thr Asp Asn Ser Cys Lys Trp Tyr Tyr Glu Cys 290 295 caa ggt gta ttc gac gtg gcc cga tat gct tgt gca Gin Gly Val Phe Asp Val Ala Arg Tyr Ala Cys Ala 305 310 ttc aac agc gtt ctg cag cag tgc gta cct gca tac Phe Asn Ser Val Leu Gin Gin Cys Val Pro Ala Tyr 320 325 tta ggt gca agt gtc act tct tcc cca tcc ata cca Leu Gly Ala Ser Val Thr Ser Ser Pro Ser Ile Pro 335 340 345 tta cca tct ttc cca acc ttg cca aca tct tcc cca Leu Pro Ser Phe Pro Thr Leu Pro Thr Ser Ser Pro 350 355 360 cct ttt ggc cga. aaa tct ctt gat atg caa. aca aaa Pro Phe Gly Arg Lys Ser Leu Asp Met Gin Thr Lys 370 375 tgt aca aaa gga gaa gtt tct aaa gac gat tta aat Cys Thr Lys Gly Glu Val Ser Lys Asp Asp Leu Asn 385 390 tagtgagtgt gttatatgaa attgctattg catattacat gttat ggtgatttat ttcatttaaa ggcaatgtat cggtttaatg tttat ttaatattaa atagattaat taaaaatcta taaagtgata ataat attattgaat attaagaagt gacggataga tctgataatg acaat ttacttaata attatgagtg gcatcattat gcaatgattt acaagcttaa 1436 gttttaaatt 1496 agaggcctga 1556 tagtagttgg 1616 atcactatta 1676 taaag tattt tatcc tatag tttattcaac attttattta actgcttgca acttttaata aaacgcatnt 31 tttattgttt taagtataaa tcttattagg gcacaatatg aaaataaaaa taaagaactt 1736 tatacaaaag ctnittttatc aatatgcttc ttgcgctatt aagttaagaa attttattta CK1 1796 tgctgggaaa aaancaanaa atgctttatt tattct 1832 00 <210> 43 <211> 397 <212> PRT <213> Ctenocephalides felis <223> At locations 3, 1675, 1749, 1810 and 1814, n c-i unknown <400> 43 Met Lys Leu Leu Leu Val Leu Leu Ala Thr Phe Ser Cys Thr Leu Ser 1 5 10 Leu Glu Ser Gin Glu Leu Ile Pro Val Glu Thr Asn Ser Ser Ser Val 25 Arg Ile Trp Gin Gin Pro Phe Ile Cys Pro Arg Val Gly Leu Phe Arg 40 Asp Pro Leu Asp Pro Thr Cys Lys Arg Tyr Tyr Lys Cys Lys Leu Gly 55 Leu Asn Gly Ala Leu Gin Ser Gly His Phe Leu Cys Pro Trp Asn Leu 70 75 Tyr Ile Ser Giu Phe Tyr Gin Arg Cys Vai Gly Pro Thr Tyr Ser Glu 90 Cys Leu Ile Thr Pro Leu Pro Pro Pro Ile Val Pro Thr Thr Thr Thr 100 105 110 Thr Thr Thr Thr Pro Ala Pro Pro Pro Leu Phe Ser Cys Val Gin Glu 115 120 125 Gly Met Phe Ile Asp Pro Tyr Asp Ser Thr Cys Lys Gly Tyr Tyr Lys 130 135 140 Cys Ala Leu Lys Ala Gly Gly Gly Phe Ser Val Ala Arg Tyr Asn Cys 145 150 155 160 Pro Gly Ser Thr Tyr Phe Ser Ser Thr Tyr Gin Gin Cys Val Val Ala 165 170 175 Ser Leu Ser Glu. Cys Leu Arg Asn Pro Pro Ala Pro Trp Gln Pro Pro 180 185 190 Thr Pro Pro Pro Ile Tyr Asn Cys Val Gin Ser Gly Ser Phe Ala Asp 195 200 205 Pro Ala Asp Lys Thr Cys Lys Val Tyr Phe Glu Cys Val Arg Leu Ser 210 215 220 Gly Gly Asn Phe Asn Val Gly Arg Phe Thr Cys Ala Gly Gin Thr Tyr 00 225 Phe Ser Ala Leu Leu Ala Thr Ala 260 Gin Pro Phe Ala 275 230 Tyr Gin 245 Gin Cys Val Ala Pro Leu Ser Glu Cys 255 Ala Pro Pro Pro Pro 265 Pro Pro Gly Pro Gly Pro Ser 270 Tyr Thr Asp Cys Val Arg Thr 280 Gly Leu Phe Leu Asp 285 Asn Ser 290 Cys Lys Trp Tyr Tyr 295 Glu Cys Thr Leu Ala Gin Gly Val Phe 305 Asp Val Ala Arg Tyr 310 Ala Cys Ala Ala Glu 315 Leu Tyr Phe Asn Ser 320 Val Leu Gin Gin Val Pro Ala Tyr Gin 330 Ser Asp Cys Leu Gly Ala 335 Ser Val Thr Phe Pro Thr 355 Ser 340 Ser Pro Ser Ile Pro 345 Ser Leu Pro Ser Leu Pro Ser 350 Pro Phe Gly Leu Pro Thr Ser Pro Thr Ala Gly Phe 365 Arg Lys 370 Ser Leu Asp Met Gin 375 Thr Lys Thr Gly Thr 380 Lys Cys Thr Lys Gly 385 Glu Val Ser Lys Asp 390 Asp Leu Asn Ser Thr Cys Thr 395 <210> 44 <211> 1832 <212> DNA <213> Ctenocephaliles felis <220> <223> At locations 19, 23, 84, 158 and 1830, n unknown <400> 44 agaataaata gcgcaagaag ttgtgcccta agcagt taaa cataattatt cttaatattc atctatttaa aatgaaataa tataacacac tacatttagt ctgttgggga atggggaaga gcagaacgct cttgagcgtt acaatcctgt gaggtgcagc cgctgaagta 1020 aagcatttnt catattgata ataagattta taaaatgttg aagtaaattg aataatatta tattaaataa atcaccataa tcactaggta tccagttttt agatgttggc agtgacactt gttgaaatat taatgtgcac ccgcacgcaa tgtcgccaag tgtctgtccg tgnttttttc aaanagcttt tacttaaaac aataaataat tctataccaa tggatatcag aaaataaatt cctttattaa catgtagaat gtttgcatat aaggttggga gcacctaaac aactcagctg tcgtagtacc gcaaagggtt cac tcggata gcgcatgtaa. ccagcataaa tgtataaagt aataaaanat agtgataaat ctactacatt gcctcttatc taaaaccatt gcttgtatgt ttaaatcgtc caagagattt aagatggtaa agtctgactg cacaagcata atttgcaaga gcgatggtcc aaggcgcttg aacgacccac taaaatttct tctttatttt gcgttttatt cattgcataa atcagatcta actttataga aaaccgatac aatatgcaat tttagaaact tcggccaaaa ggatggtaag gtatgcaggt tcgggccacg attatcagtg tggtcctggt cacgcattgt gttgaaattt taacttaata tattttcata aaaagttgca tgatgccact tccgtcactt tttttaatta attgccttta agcaatttca tctccttttg ggaaatcc tg gatggtatgg acgcactgct tcgaatacac taatccaaaa ggcggtggtg t ggt aa agog cctccagaga 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 00 gtctcacaca 1080 tctgcacgca 1140 ttaggcactc 1200 aaccaggaca 1260 agtatccttt 1320 aaagaggcgg 1380 gcagaggtgt 1440 ttatatacaa 1500 gtttgcactt 1560 ttggacaaat 1620 ttagttcttg 1680 gtttcatcgt 1740 taatgatatt 1800 ccactgcttg 1832 ttcaaaatat gttgtatatg actttgcatg ttttgtctgc tggatctgca aatgagccgc ggcggcggcg taggtggttg ccaaggcgct ggaggatttc cgaaagggaa gcgaccacac attgctgata cgtgctgctg aagtaagtag attgtaacgc gctacactga atcctccgcc tgctttcaaa gcacatttgt gcaagtgctg Lcatagggat ctataaacat cccttcctgc acgcatgaaa cggtgctggt gtagttgtcg tagtcgtagt agtcggaaca attggaggag aattaaacat tccgaatatg tagggcccac acatctttgg taaaactcac gttccatggg cataaaaaat gaccgctttg cagagcacca tttagaccaa gtagtacctt ttgcatgtag gatccaatgg atcgcgaaat agtccaactc gaatggttgc tgccaaatac gaacgctgct ggaatttgtt tcaactggaa agactccaaa gacaatgtac acgagaaggt agccaagagc actaaaagca aatgaaacgc gatttaacag attccacttt aaaataaaca atttttttga gtaccaaaaa tttgaagtta ctaaatcccc cgcgtactct gcgttgttac ccctatagtg agtcgtattn ga <210> <211> 1191 <212> DNA <213> Ctenocephalides fells <400> atgaaactgc gaactaattc tgtccaagag tgcaaacttg tatataagtg cctctgcctc cctctttttt ggatac taca cctggttcta tgcctaagaa gtgcagagcg gtgagactct ttcagcgcgc gcacctccac ggattgtttt gctcaaggtg gttctgcagc 1020 tccccatcca 1080 ccaacagcag 1140 aaatgtacaa 1191 ttttagtgct cagttgaaac ttggactatt gtctaaatgg agttttacca ctccaattgt catgcgtgca aatgtgcttt cttacttcag atcctccagc gctcatttgc ctggaggaaa tttaccaaca caccgccacc tggattacac tattcgacgt agtgcgtacc cttggctacc aaattccagc t cgc gat cc a tgctctgcaa aagatgtgtg tccgactact ggaagggatg gaaagcaggc cagcacgtat gccttggcaa agatccagca tttcaacgtg atgcgtgcaa aggaccagga tgataattct ggcccgatat tgcataccag ttctcgtgta agcgttcgta ttggatccta agcggtcatt ggccctacat- acgactacga t ttatagat c ggaggat tca cagcaatgtg ccacctacgc gacaaaacat ggtcgtttta gcgcctttat ccatcgcaac tgcaaatggt gcttgtgcag tcagactgtt cattgtcttt t ttggcagca catgcaaaag ttttatgccc attcggaatg caac tacacc cctatgacag gtgtagcgcg tggtcgcttc cgccgcccat gcaaagtata catgcgccgg ccgagtgctt cctttgcttg actacgagtg ctgagttata taggtgcaag ggagtctcaa accattcatt gtactacaag atggaacttg tttaattaca agcaccgccg cacttgcaaa ttacaattgt cctttcggag atacaactgc ttttgaatgt acagacatac ggcgacagct cgtgcggaca cacattaaac tttcaacagc tgtcacttct taccatcctt accatcctta ccatctttcc caaccttgcc aacatcttcc gatttccttt tggccgaaaa tctcttgata tgcaaacaaa aactggaact aaggagaagt ttctaaagac gatttaaatt ctacatgtac c 00 <210> 46 <211> 1191 <212> DNA <213> Cterxocephalides felis <400> 46 ggtacatgta ttttgtttgC tggcaaggtt acttgcacct atataac tca gcactcgtag gcaagcaaag caagcactcg tccggcgcat atatactttg tatgggcggC ggaagcgacc acgcgctaca gctgtcatag tggtgtagtt ac at t ccgaa tgggcataaa 1020 cc t tttgcat 1080 ttgctgccaa 1140 caaagacaat 1191 gaatttaaat ata tcaagag gggaaagatg aaacagtctg gctgcacaag taccatttgc ggttgcgatg gataaaggcg gtaaaacgac catgttttgt ggcgtaggtg acacattgct ctgaatcctc ggatctataa gtcgtagtcg tatgtagggc aaatgaccgc cgtctttaga at t ttcggcc gtaaggatgg actggtatgc catatcgggc aagaattatc gtcctggtCC cttgcacgca ccacgttgaa ctgctggatc gttgccaagg gatacgtgct cgcctgcttt acatcccttc tagtagtcgg ccacacatct tttgcagagc aacttctcct aaaaggaaat taaggatggt aggtacgcac cacgtcgaat agtgtaatcc tggtggcggt ttgttggtaa atttcctcca tgcaaatgag cgctggagga gc tgaagtaa caaagcacat ctgcacgcat aacaattgga. ttggtaaaac accatttaga tttgtacatt cctgctgttg atggatgggg tgctgcagaa acaccttgag aaaaacaatc ggtggaggtg agcgcgctga gagagtctca ccgctctgca tttcttaggc gtagaaccag ttgtagtatc gaaaaaagag ggaggcagag tcacttatat. ccaagtttgc tagttccagt gggaagatgt aagaagtgac cgctgttgaa cgtttaatgt c tgtccgcac cagctgtcgc agtatgtc tg cacattcaaa cgcagttgta actccgaaag gacaattgta ctttgcaagt gcggcggtgc gtgtaattaa acaagttcca acttgtagta 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 gtaggatcca atggatcgcg aaatagtcca actcttggac aaatgaatgg atacgaacgc tgctggaatt tgtttcaact ggaattagtt cttgagactc gtacacgaga aggtagccaa gagcactaaa agcagtttca t <210> 47 <211> 1161 <212> DNA <213> Ctenocephalides felis <220> <221> CDS <222> (1)..(1143) <400> 47 ttg gag tct caa gaa cta att cca gtt Leu Gin Ser Gin Gin Leu Ile Pro Val 1 5 gaa Glu 10 aca aat tcc agc Thr Asn Ser Ser agc gtt Ser Val cgt att. tgg Arg Ile Trp gat cca. ttg Asp Pro Leu caa cca ttc att Gin Pro Phe Ile cca aga gtt gga Pro Arg Val Gly cta ttt cgc Len Phe Arg aaa ctt ggt Lys Len Gly gat cct aca tgc Asp Pro Thr Cys aaa Lys agg tac tac aag Arg Tyr Tyr Lys cta aat Leu Asn ggt gct ctg caa Giy Ala Leu Gin ggt cat ttt tta Gly His Phe Len tgC Cys cca tgg aac ttg Pro Trp Asn Len tat ata. agt gag ttt tac caa aga tgt gtg ggc cct aca tat tcg gaa Tyr Ile Ser Gin Phe Tyr Gin Arg Cys Val Gly Pro Thr Tyr Ser Gin tgt tta att aca cct ctg cct cct cca Cys Leu Ile Thr Pro Leu Pro Pro Pro gtt ccg act act acg act Val Pro Thr Thr Thr Thr 00 288 336 acg aca act Thr Thr Thr ggg atg ttt Gly Met Phe 115 aca Thr 100 cca gca ccg ccg cct ctt ttt tca tgc Pro Ala Pro Pro Pro Leu Phe Ser Cys 105 gtg cag gaa Val Gln Glu 110 tac tac aaa Tyr Tyr Lys ata gat ccc tat Ile Asp Pro Tyr gac Asp 120 aga act tgc aaa Ser Thr Cys Lys gga Gly 125 tgt gct Cys Ala 130 ttg aaa. gca ggc Leu Lys Ala Gly gga ttc agt gta gcg cgt tac aat tgt Gly Phe Ser Val Ala Arg Tyr Asn Cys 140 cct Pro 145 ggt tct act tac Gly Ser Thr Tyr t ta Phe 150 agc aga acg tat Ser Ser Thr Tyr cag Gin 155 caa tgt gtg gtc Gin Cys Val Val gat Ala 160 480 528 tcc ctt tcg gag Ser Leu Ser Gin tgc Cys 165 cta aga aat cat Leu Arg Asn Pro gcg cct tgg caa Ala Pro Trp Gin cca cct Pro Pro 175 acg ccg ccg Thr Pro Pro cca gca gac Pro Ala Asp 195 ata tac aac tgc Ile Tyr Asn Cys cag agc ggc tca Gin Ser Gly Ser ttt gca gat Phe Ala Asp 190 aga ctc tct Arg Len Ser 576 624 aaa aca tgc aaa Lys Thr Cys Lys gta Val 200 tat ttt gaa tgt Tyr Phe Glu Cys gga gga Gly Gly 210 aat ttc aaa gtg Asn Phe Asn Val ggt cgt Gly Arg 215 ttt aca tgc Phe Thr Cys gcc Ala 220 gga cag aca tac Gly Gin Tlir Tyr ttc agc gcg ctt tac Phe Ser Ala Leu Tyr 225 ttg gcg aca gct gca Len Ala Thr Ala Ala 245 c aa Gin 230 caa tgc gtg caa Gin Cys Val Gin gcg Al a 235 cct tta tcc gag Pro Leu Ser Glu 672 720 768 cct cca cca ccg Pro Pro Pro Pro cca Pro 250 cca gga cca gga Pro Gly Pro Gly cca tcg Pro Ser 255 aaa ccc ttt Gin Pro Phe aat tct tgc Asn Ser Cys 275 gc t Ala 260 tgc gtg cgg aca Cys Val Arg Thr ttg ttt ttg gat Len Phe Leu Asp tac act gat Tyr Thr Asp 270 caa ggt gta Gin Gly Val 816 864 aaa tgg tac tac Lys Trp Tyr Tyr tgc aca tta aac Cys Thr Leu Asn gct Ala 285 ttc gac Phe Asp 290 gtg gcc cga tat Val Ala Arg Tyr gc t Al a 295 tgt gca gct gag Cys Ala Ala Glu tta Leu 300 tat ttc aac agc Tyr Phe Asn Ser gtt Val 305 ctg cag cag tgc gta act gca tac cag Len Gin Gin Cys Val Pro Ala Tyr Gin tca Ser 315 gac tgt tta ggt Asp Cys Leu Gly gca Ala 320 960 00 agt gtc 1008 Ser Val ttc coa 1056 Phe Pro act tct Thr Ser acc ttg Thr Leu 340 tcc Ser tot Ser cga aaa tct ott gat atg caa 1104 Arg Lys Ser Leu Asp Met Gin 355 gga gaa gtt tct aaa gao gat 1153 Gly Glu Val Ser Lys Asp Asp 370 375 gttatatg 1161 <210> 48 <211> 381 <212> PRT <213> Ctenocephalides felis <400> 48 Leu Giu Ser Gin Giu Leu Ile 1 5 Arg Ile Trp Gin Gin Pro Phe Asp Pro Leu Asp Pro Thr Cys Leu Asn Gly Ala Leu Gin Ser 55 Tyr Ile Ser Glu Phe Tyr Gin 70 Cys Leu Ile Thr Pro Leu Pro Thr Thr Thr Thr Pro Ala Pro 100 Gly Met Phe Ile Asp Pro Tyr 115 Cys Ala Leu Lys Ala Gly Gly 130 135 ata Ile too Ser ao a Thr 360 tta Leu Pro Ile Lys 40 Gly Arg Pro Pro Asp 120 Gly oca too Pro Ser 330 oca aoa Pro Thr 345 aaa aot Lys Thr aat tot Asn Ser Val Cys 25 Arg His Cys Pro Pro 105 S er Phe Glu Pro Tyr Phe Val Ile Leu Thr Ser gca Al a gga Giy aoa Thr Thr Arg Tyr Leu Gly 75 Val Phe Cys Val1 tta oca too Theu Pro Ser gga Gly aoct Thr tgt Cys 380 Asn Val Lys Cys Pro Pro Ser Lys Ala 140 aaa Lys 365 acc Thr Ser Gly Cys Pro Thr Thr Cys Gly 125 Arg ttt Phe tta oca tot Leu Pro Ser 335 cot ttt ggo Pro Phe Gly 350 tgt aoa aaa Cys Thr Lys tagtgagtgt Ser Leu Lys Trp, Tyr Thr Val 110 Tyr Tyr Val1 Arg Gly Leu Giu Thr Giu Lys Cys Pro 145 Gly Ser Thr Tyr Phe 150 Ser Ser Thr Tyr Gin Cys Val Val Ser Leu Ser Giu Cys Leu Arg 165 Thr Pro Pro Pro Ile Tyr Asn 180 Asn Pro Pro Ala Pro Trp Gin Pro Pro 175 Gin Ser Gly Ser Phe Ala Asp 190 Pro Aia Asp*Lys 195 Thr Cys Lys Vai 200 Tyr Phe Glu Cys Arg Leu Ser Giy Giy 210 Asn Phe Asn Val Arg Phe Thr Cys Aia 220 Gly Gin Thr Tyr Phe 225 Ser Ala Leu Tyr Gin 230 Gin Cys Val Gin Pro Leu Ser Giu Cys 240 Leu Ala Thr Ala Ala 245 Pro Pro Pro Pro Pro Gly Pro Gly Pro Ser 255 Gin Pro Phe Asn Ser Cys 275 Aila 260 Cys Val Arg Thr Leu Phe Leu Asp Tyr Thr Asp 270 Gin Gly Val Lys Trp Tyr Tyr Giu 280 Cys Thr Leu Asn Al a 285 Phe Asp 290 Val Ala Arg Tyr Aia 295 Cys Ala Ala Glu Leu 300 Tyr Phe Asn Ser Val 305 Leu Gin Gin Cys Pro Ala Tyr Gin Asp Cys Leu Gly Ser Val Thr Ser Pro Ser Ile Pro Ser 330 Leu Pro Ser Leu Pro Ser 335 Phe Pro Thr Arg Lys Ser 355 Gly Giu Vai 370 Leu 340 Pro Thr Ser Ser Thr Ala Gly Phe Pro Phe Gly 350 Cys Thr Lys Leu Asp Met Gin Thr 360 Lys Thr Gly Thr Lys 365 Ser Lys Asp Asp 375 Leu Asn Ser Thr Cys Thr 380 <210> 49 <211> 1161 <212> DNA <213> Ctenocephalides felis <400> 49 catataacac tgtacattta tgctgttggg ggatggggaa ctgcagaacg accttgagcg aaacaatcct tggaggtgca cgcgctgaag gagtctcaca gctctgcacg actcactagg gttccagttt gaagatgttg gaagtgacac ctgttgaaat tttaatgtgc gtccgcacgc gctgtcgcca taigtctgtc cattcaaaat cagttgtata tacatgtaga atttaaatcg tctttagaaa cttctccttt ttgtttgcat gcaaggttgg ttgcacctaa ataactcagc actcgtagta aagcaaaggg agcactcgga cggcgcatgt atactttgca tgggcggcgg atcaagagat gaaagatggt acagtctgac tgcacaagca ccatttgcaa ttgcgatggt taaaggcgct aaaacgaccc tgttttgtct cgtaggtggt tttcggccaa aaggatggta tggtatgcag tatcgggcca gaattatcag cctggtcctg tgcacgcatt acgttgaaat gctggatctg tgccaaggcg aaggaaatcc aggatggtat gtacgcactg cgtcgaatac tgtaatccaa gtggcggtgg gttggtaaag ttcctccaga caaatgagcc ctggaggatt 120 180 240 300 360 420 480 540 600 660 tcttaggcac tccgaaaggg aagcgaccac 00 agaaccagga gtagtatcct aaaaagaggc aggcag-aggt acttatatac 1020 aagtttgcac 1080 tcttggacaa 1140 aattagttct 1161 caattgtaac ttgcaagtgc ggcggtgctg gtaattaaac aagttccatg gcgctacact tgtcataggg gtgtagttgt attccgaata ggcataaaaa acattgctga gaatcctccg atctataaac cgtagtcgta tgtagggccc atgaccgctt tacgtgctgc cctgctttca atcccttcct gtagtccsgaa acacatcttt tgcagagcac t gaagtaagt aagcacattt gcacgcatga caattggagg ggtaaaactc catttagacc 720 780 840 900 960 ttgtagtacc ttttgcatgt aggatccaat ggatcgcgaa atagtccaac atgaatggtt gctgccaaat acgaacgctg ctggaatttg tttcaactgg tgagactcca a <210> <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer Synthetic <400> cgggatcctg ctgacaggaa ttcgcccac <210> <211> <212> <213> 51 31 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 51 catggtaccc ctggtttaag ccttacttag c Synthetic <210> 52 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 52 gtctggaagc tcaggaagag g <210> 53 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Synthetic 00 CN Primer <400> 53 ccatcctaat acgactcact atagggc <210> 54 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Primer <400> 54 actcactata gggctcgagc ggc <210> <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer Synthetic <400> gtaatatgcg tgacaatcgt gtgg <210> 56 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer Synthetic <400> 56 cggtgcaagt tatagaacct tccg <210> 57 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 57 cgggatcccg aatatgctga cgtagatgtg tg Synthetic <210> 58 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer Synthetic <400> 58 ggaattctgt tttattctgg ttggtaacat tc <210> 59 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Primer <400> 59 gatatccact ttgatcagcg cac <210> <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Primer <400> ggtactactc ctggtgcggg c <210> <211> <212> <213> 61 23 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Primer Synthetic <400> 61 ccgtcgacat taaactcacc atc <210> 62 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 62 cgggatcctt atgatggtga gtttaatgtc g Synthetic <210> 63 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 63 ggggtacctt aatataattt aggtttcctc tcgc <210> 64 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 64 gcgcatgtaa aacgacccac g <210> <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer Synthetic Synthetic Synthetic <400> ctaatacgac tcactatagg gcaagcagtg gtaacaacgc agagt <210> 66 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer Synthetic <400> 66 ctaatacgac tcactatagg gc <210> 67 <211> <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Primer 42 <400> 67 cgggtacctt ggagtctcaa gaactaattc <210> 68 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic Primer <400> 68 aggaattcca tataacacac tcactaggta catgtag 37