MXPA99011678A - Receptor for a bacillus thuringiensis toxin - Google Patents

Abstract

The cDNA that encodes a glycoprotein receptor from the tobacco hornworm which binds a i(Bacillus thuringiensis) toxin has been obtained and sequenced. The availability of this cDNA permits the retrieval of DNAs encoding homologous receptors in other insects and organisms as well as the design of assays for the cytotoxicity and binding affinity of potential pesticides and the development of methods to manipulate natural and/or introduced homologous receptors and, thus, to destroy target cells, tissues and/or organisms.

Description

f * - 25 -, * Brief Description of the Invention The present invention is based, in part, on the isolation and characterization of a receptor that binds with members of the BT toxin family of insecticidal proteins, hence forward the BT-Ri protein. The present invention is also based on the isolation and characterization of a molecule of phyclic acid that encodes the receptor of the BT-toxin, henceforth BT-R gene ?. Based on these observations, the present invention provides compositions and methods for the use of CT identify agents that bind BT-Ri protein as a means to identify the insecticidal agent and to identify other members of the BT-R protein family. ? BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 (SEQ ID NOS: 1-2) shows the ucleucleotide sequence and the amino acid sequence of inferred ADNC encoding the BT-Ri protein of the M. sixth. Figure 2 (panels a and b) (SEQ ID NOS: 2) shows a comparison of the sequences of .t anino acids of cadherin motifs (BTRcad-1 to 11) in BT- 25 Rj.with others of cadherins.

Figure 3 (SEQ ID NOS: 3-17) shows a block diagram of the structure similar to the cadherin of BT-R ?. * 5 Figure 4 shows the cloning characterization of the BamHI-SacI fragment of BT-Ri. LM, that's the HindIII cut that Lambda makes; UP is the uncut plasmid clone; NP is the plasmid cut Nsil; XP is the Xhol plasmid cut; BSP is the plasmid cut BamHl and Sacl that show & 1 cloned fragment of BT-Ri; RM is RNA, size arc; and RT1 and RT2 are transcribed as mRNA of the cloned BT-Ri fragment. Figure 5 illustrates the detection of the expression of the plasmid protein containing the Bam-Sac fragment of BT-R? using the 3 S-methionine as a brand. LCR is an mRNA for the control of luciferase that shows the reticulocyte rabbit lysates are functional; 1 and RR2 are products of expression of the Bam-Sac fragment of BT-R? produced in the rabbit reticulocytes of the mRNA; LCT is a plasmid for the control of luciferase that shows that the set of functional transcription / conversion; and TT1 and fí > w -T.T2 are expression products of the fragment of .1? BT-Ri produced in the transcription / translation set. Figure 6 shows a radio-blot of the Bam-Sac fragment of BT-Ri, with 125-labeled CrylAb. BBMV are the membrane vesicles that abut the weeds of the middle part of the intestine of M. sex ta that contain the wild type BT-Ri receptor protein; RBK is a target rabbit reticulocyte 10; and RR1 and RR2 are the expression products of the Bam-Sac fragment of BT-Ri produced in the rabbit reticulocytes of the mRNA; TBK is a target transcription / conversion set; TT1 and TT2 are 15 expression products of the Bam-Sac fragment of BT-Ri produced in the transcription / translation set. The arrows point to two of the bands. Figure 7 shows the presence of a BT-Ri homologue in the corn moth larva and the identified corn borer larvae using the binding similar to that used to identify the original BT-Ri clone. Figure 8 shows the binding of Crylab to fragments of the protein BT-R ?. ?yes Detailed Description of the Invention M I. General Description The present invention is based, in part, on the isolation and characterization of a novel protein expressed in the middle part of the sixth mandibular intestine that binds the family members BT-toxin proteins, 10 hereby forward the BT-R protein ?. The present invention specifically provides BT-R? purified, the amino acid sequence of BT- _ JEti, as well as the nucleotide sequence encoding BT-Ri. The protein molecules BT-Rx 15 and nucleic acid can serve as targets when identifying insecticidal agents.

II. Specific Modalities A. Protein BT-R? They were known before the present invention, although members of the BT-toxin protein family have not identified any receptor that binds these proteins to toxins. The present invention provides, in part, the amino acid sequence of a BT-toxin receptor that manifests in the mid-intestine of Ma du ca sixth.

In one embodiment, the present invention provides the ability to isolate or produce a previously unknown protein by using purification methods, the cloned nucleic acid molecules described herein or by synthesizing a protein having the amino acid sequence set forth herein. F- As used herein, BT-Ri refers to one - > protein having the BT-Ri amino acid sequence provided in Figure 1, as well as allelic variants of the BT-Ri sequence, and mutants with conservative substitutions of the BT-Ri, sequence having BT-Ri activity. ST-R? it is comprised of a simple subunit, has a molecular weight of 210 kD, and has the amino acid sequence provided in Figure 1. A prediction of the BT-Ri structure is provided in Figure 3.

The BT-R protein? of the present invention includes the specifically identified and characterized variant described herein, as well as allelic variants, variants with conservative substitution and homologs (Figure 7) which can be isolated / generated and characterized without inappropriate experimentation following the methods outlined below. For convenience, all BT-Ri proteins will collectively be referred to as the BT-R proteins, the BT-Ri proteins of the present invention or BT-10 Ri.

The term BT-Ri includes all the naturally occurring allelic variations of the BT-Ri protein of Ma indu ca sixth provided in Figure 1. In general, the naturally occurring allelic variations of the BT-Ri of the Mandu ca sixth they will share significant homology, at least 75% and generally at least 90%, with the amino acid sequence of 20 BT-Ri provided in Sec ID No .: 2. The allelic variants, however, have a slight difference in the amino acid sequence that Sec. ID No .: 2, will be expressed as a transmembrane protein in the digestive tract 25 of an insect or other organism. Typically, * -. allelic variations of the BT-Ri protein will contain moderate amino acid substitutions in the BT-Ri sequence described herein or will contain a substitution of an amino acid at a position in the BT-Ri homologue (a BT-Ri protein isolated from an organism instead of the Manduca sixth).

A class of allelic variants BT-Rj. will be the proteins that share a high degree of homology with at least a small region of the amino acid sequence provided in SEC. ID No: 1, but may also contain a radical that moves away from the sequence such as an unmoderated substitution, truncation, insertion or structure change. These alleles are terminated in some BT-Ri mutants and represent proteins that typically do not develop the same biological functions as the BT-Ri variant of SECs does. ID No: 2 The BT-Ri proteins of the present invention are preferably in isolated form. As used herein, a protein is said to be isolated when physical, mechanical or chemical methods are employed to remove the BT-Ri protein from cellular constituents that are normally associated with the protein. A person skilled in the art can easily employ standard purification methods to obtain a BT-R protein? isolated The nature and degree of isolation will depend on the intended use.

The cloning of the nucleic acid molecule encoding the BT-R? makes it possible to generate defined fragments of BT-R proteins? of the present invention. As discussed below, the fragments of BT-R? they are particularly useful in: generating field-specific antibodies; identify agents that bind to the toxin that binds the field in BT-R ?; identify toxins that bind to structures; identify cellular factors that bind BT-20 R; isolate homologues or other allelic forms of BT-R ?; and study the method of action of BT-toxins. i, 4h Fragments of proteins BT-R? can be generated using the standard peptide synthesis technology and the amino acid sequence of BT-R? Mandu ca sex ta exposed here. Alternatively as illustrated in example 5, recombinant methods 5 can be used to generate nucleic acid molecules encoding a fragment of the BT-R protein ?. Fragments of the BT-Ex protein subunits containing the structures of particular interest can be identified using methods known in the art such as by using an immunogenicity label, the Chou.Fasman brand, 4. the Garnier-Robson brand, the brand 'Kyte- Doolittle, the Eisenberg brand, the Karplus- Schultz brand or the Jameson-Wolf brand of the 15 BT-R protein ?. Fragments that have these residues are particularly useful for generating anti-BT-R? of specific field or to identify cellular factors that bind to r BT-R ?. A particular fragment that is preferred for use in the identification of insecticidal agents is a soluble fragment of BT-R? which can bind a member of the B toxin family. In Example 5, a fragment of BT-R that binds to a BT-toxin is exposed. 25 * * i- As described below, the members of the BT-R protein family? can be used for, but not limited to: 1) a target to identify agents that bind BT-R ?, 2) a target or a hook to identify and isolate the binding partners and the cellular factors that bind to BT -R ?, 3) a test target to identify BT-R? and another receptor-mediating activity, and 4) a cell marker that signals a member of the BT-Ri protein family.

B. Anti-BT-R antibodies? The present invention also provides antibodies that bind BT-R ?. The most preferred antibodies will selectively bind to the BT-Rx and will not bind (or only weakly bind) to non-BT-R proteins. Anti-BT-R? which are especially contemplated include the monoclonal and polyclonal antibodies as well as the fragments containing the antigen that binds to the field and / or one or more complements that determine the regions (CDRs) of these antibodies.

Antibodies are generally prepared by immunizing a suitable mammalian host using a BT-R protein? (synthetic or isolated), or fragment, in isolated or immunoconjugated form (Harlow, Antibodies, Cold, uta Bpring Harbor Press, NY (1989)). The regions of the protein BT-R? that show immunogenic structure can be easily identified %. 10 using the methods known in the art. Other regions and important fields may J is easily identified using the analytical and comparative methods of the protein known in the art, such as the Chou.Fasman analysis, 15 Garnier-Robson, Kyte-Doolittle, Eisenberg, arplus-Schultz or Jameson-Wolf. Fragments containing these residues are particularly suitable for generating specific classes of anti-BT-R ?. Particularly useful fragments include, but are not limited to, the BT-toxin that binds to the BT-R field? identified in Example 5.

Methods for preparing a protein for use as an immunogen and for preparing immunogenic conjugates of a protein with a carrier such as BSA, KLH, and other carrier proteins are well known in the art. In some circumstances, direct conjugation with reagents such as carbodiimide may be used; in other circumstances binding reagents such as those provided by Pierce Chemical Co., Rockford, IL, can be effective.

The administration of an immunogen BT-R? it is generally performed by injection for a suitable period of time in combination with a suitable adjuvant, as is generally understood in the art. During the immunization schedule, the validity of antibodies that can be taken to determine the adequate formation of the antibody.

Although the polyclonal antiserum produced in this way may be satisfactory for some applications, for other applications, monoclonal antibody preparations are preferred. Branches of immortalized cells that secrete a desired monoclonal antibody can be prepared using the standard Kohier and Milstein method or modifications where the immortalization effect of lymphocytes or spleen cells is generally known. The derivations of immortalized cells that secrete the desired antibodies are projected by immunoassay where the antigen is the protein BT-R? or the BT-R ?. When the culture of the appropriate immunized cell that secretes the desired antibody is identified, the cells can be cultured in vi tro or by production of serous fluids.

The desired monoclonal antibodies are then recovered from the culture supernatant or the serous supernatant. The fragments of the monoclonal or polyclonal antiserum containing the immunologically significant portion can be used as antagonists, as well as the intact antibodies. The use of immunologically reactive fragments, such as Fab, Fab ', F (ab') 2 fragments, is frequently preferred, especially in a therapeutic context, since these fragments are generally less immunogenic than all immunoglobulin.

Antibodies or fragments can also be produced, using current technology, by recombinant means. The regions that bind specifically to the desired regions of the BT-R protein? they can also be produced in the context of chimeric antibodies or grafted with CDRs of multiple species origin.

As described below, the anti-BT-R? are useful as modulators of BT-R? activity, are useful in anti-viral antibodies based on assay methods to detect BT-R? expression / activity by generating conjugated toxins, to purify BT-R? of Mandu ca sex ta, when generating anti-ideotypic antibodies that mimic the BT-R protein? and identify competitive inhibitors of toxin-BT / BT-R interactions ?.

C. Nucleic Acid Molecules that Code BT-R? As described above, the present invention is based, in part, on isolating Manduca sexta nucleic acid molecules encoding the BT-R ?. Accordingly, the present invention also provides nucleic acid molecules encoding the BT-Rα protein, as defined herein, preferably in isolated form. For convenience, the nucleic acid molecules encoding the BT-R? will they be referred to as nucleic acid molecules encoding BT-R ?, the B T-Rl f or B T-R genes? . The nucleotide sequence of the Manduca sexta nucleic acid molecule encoding an allelic form of the BT-R ?.

As used herein, a "nucleic acid molecule" is defined as an RNA or DNA molecule that encodes a peptide that has been previously defined, or is in addition to a nucleic acid sequence encoding these peptides. Particularly preferred nucleic acid molecules will have a nucleotide sequence identical or complementary to the Manduca s DNA sequences set forth herein. Especially contemplated are genomic DNA, cDNA, synthetically prepared DNA, and reverse-sense molecules, as well as nucleic acids based on an alternating chain or include alternate bases, if derived from natural or synthesized sources. A person skilled in the art will readily obtain these classes of nucleic acid molecules using the BT-R? described here. However, these nucleic acid molecules are further defined to be novel and are not obvious on any of the nucleic acid molecules of the prior art encoding non-BT-R proteins ?. For example, the sequences of the present invention specifically exclude previously identified nucleic acid molecules that share only partial homology with BT-R ?. These excluded sequences include identified members of the cadherin family of proteins.

As used herein, a nucleic acid molecule is said to be "isolated" when the nucleic acid molecule is substantially separated from the contaminating nucleic acid molecules encoding the peptides instead of the BT-R ?. A person skilled in the art can easily employ nucleic acid isolation methods to obtain a nucleic acid molecule that encodes BT-R? isolated The present invention also provides fragments of the nucleic acid molecules encoding BT-R? of the present invention. As used herein, a fragment of a nucleic acid molecule that encodes BT-R refers to a small portion of the entire BT-R ?. The size of the fragment will be determined by its projected use. For example, if the fragment is chosen so that it codes for the toxin that binds to the BT-R field? identified in Example 5, then the fragment will need to be long enough to encode the toxin that binds to the BT-R ?. If the fragment is used as a nucleic acid test or PCR primer, then the length of the fragment is chosen in order to obtain a relatively small number of false positives during the test / prime. The fragments of the BT-R? of Mandu ca sixth that are particularly useful as evidence of * _ Selective hybridization or PCR primers can be easily identified from the entire BT-R sequence? using methods known in the art.

Another class of fragments of nucleic acid molecules encoding BT-R? are the labeling control sequences found upstream and downstream of the region found in the genomic clones encoding the BT-R? of the BT-R gene ?. Specifically, the tissue and the specific marking control elements of development can be identified that are 5 'in the region found in the genomic clones that the BT-R encodes? of the BT-R gene ?. These labeling control sequences are useful for generating labeling vectors to label genes in the digestive tract of a transgenic organism. As described in more detail below, a person skilled in the art can easily use the BT-R cDNA sequence? described here to isolate and identify the BT-R sequences? genomic and control dialing elements found in the BT-Rj gene.

The fragments of the nucleic acid molecules that encode the BT-R? of the present invention (for example synthetic oligonucleotides) which are used as specific tests or primers for the polymerase chain reaction (PCR), or to synthesize sequences of the gene encoding the BT-R ?, proteins can be easily synthesized by techniques chemical, for example, the method fosfotrieter Matteucci, et al, J Am Chem Soc (1981) 103:.... 3185-3191, or using automated synthesis methods. In addition, longer DNA segments can be easily prepared by well-known methods, such as the synthesis of a group of oligonucleotides that define several modular segments of the BT-R ?, gene followed by the linking of oligonucleotides to create the BT-R gene. ? completely modified.

The nucleic acid molecules encoding the BT-R? of the present invention may further be modified so as to contain a detectable label for the purposes of diagnosis and testing. As described above these tests can be used to identify nucleic acid molecules that encode other allelic or homologous variations of the BT-R? and as described below these tests can be used to identify the presence of a BT-R protein? as a means to identify cells that label a BT-R protein ?. A variety of these marks are known in the art and can be easily employed with the molecules that BT-R encodes? described here. Suitable labels include, but are not limited to, biodine, radiolabeled biotin nucleotides and the like. A person skilled in the art can employ any of the art-known brands to obtain a labeled nucleic acid molecule that encodes BT-R ?.

D. Isolation of other Nucleic Acid Molecules that Code BT-R? The identification of the BT-R protein? of Mandu ca sixth and the corresponding coding with nucleic acid molecules, has made possible the identification and isolation of: 1) BT-R proteins? of organisms instead of Mandu ca sex ta, hereinafter referred to collectively as BT-R? homologs, 2) other allelic and mutant forms of the BT-R protein? Mandu ca sex ta (described above), and 3) the corresponding genomic DNA that contains the BT-R ?. The most preferred source of BT-R counterparts? they are insects, the most preferred insects are members of the order Lepidoptero, Coleoptera and Diptero. The evidence of the existence of BT-R? Homologs is provided in Figure 7.

Essentially, a person with experience in the art can easily use the amino acid sequence of the BT-R protein? Manduca sixth to generate antibody tests to project a pattern of prepared tags of cells and organisms. Typically, polyclonal antiserum of mammals such as rabbits immunized with the purified protein (as described above) or monoclonal antibodies can be used to test a tag pattern, prepared from a target organism, to obtain the coding sequence appropriate for a BT-R counterpart? The cloned cDNA sequence can be expressed as a fusion of the protein, expressed directly using its own control sequences, or tagged to construct a tag record using appropriate control sequences in a particular host used for the labeling of the enzyme.

Alternatively, a portion of the sequence encoding BT-R? described here can be synthesized and used as a test to recover the DNA encoding a member of the BT-R protein family? of organisms instead of Mandu ca sex ta, the allelic variants of the BT-R protein? of Mandu ca sixth described here, and the genomic sequence that contains the BT-R ?. Oligomers containing about 18-20 nucleotides (encoding about a 6-7 amino acid line) are prepared and used to project genomic DNA and cDNA patterns to obtain hybridization under stringent conditions or conditions of sufficient stringency to eliminate inappropriate level of false positives.

Additionally, pairs of oligonucleotide primers can be prepared for use in a polymerase chain reaction (PCR) to selectively amplify / clone a nucleic acid molecule encoding the BT-Rx, or fragment thereof. A PCR denaturing / composing / extending the cycle to use the PCR primers is well known in the art and can easily be adapted when used to isolate other nucleic acid molecules encoding BT-R ?. The regions of the BT-R? which is particularly suitable for use as a test or as a primer can be easily identified by a person skilled in the art.

The BT-R? no-Mandu ca sixth, naturally allelic variants of the BT-R gene occur? of Mandu ca sex ta and the BT-R sequences? will genomes share a high degree of homology with the BT-R sequence? of Manduca sixth described here. In general, the nucleic acid molecules will hybridize in the sequence BT-R? of Mandu ca sixth under great rigidity. These sequences will typically contain at least 70% homology, preferably at least 80%, more preferably at least 90% homology with the BT-R? of Mandu ca sixth of Sec. ID No: 1.

In general, the nucleic acid molecules that encode the BT-R? of Mandu ca sex ta will they hybridize in the BT-R sequence? of Mandu ca sixth under strict conditions. "Strict conditions" are those that (1) employ low ionic strength and high temperature to wash, for example, 0.015 M NaCl / 0.0015 M sodium titrant / 0.1% SDS at 50 ° C, or (2) used during the hybridization a denaturing agent such as formamide, for example, 50% formamide (vol / vol) with 0.1% bovine albumin serum / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42 ° C. Another example is the use of 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, Salmon sonicated (50 μg / ml), SDS 0.1%, and sulfate dextran 10% at 42 ° C, washed at 42 ° C in 0.2 x SSC and 0.1% SDS. A person skilled in the art can easily determine and vary the stiffness conditions appropriately to obtain a clear and detectable hybridization signal.

The presence of similar receptors in organisms that are not insects as well as other insects along with those that protect the BT-R? is it supported by the similar sequence of the BT-R protein? so that of the different members of the cadherin superfamily of proteins, which are membrane glycoproteins, are thought to mediate aggregation and classify the cell that depends on calcium. See, for example, Takeichi, M. Science (1991) 251: 1451; and Takeichi, M.N. Rev Biochem (1990) 50: 237.

Included in this superfamily are desmoglien, desmocollins the tumor suppressor Drosophi fa t, the protein of the intestinal peptide transport Manduca sexta and T-Cadherin. All these proteins share common extracellular standards although their cytoplasmic fields differ. Good in, L. et al. Bi ochem Bi ophys Res Commun (1990) 173: 1224; Holton, J. L. et al. J. Cel l Sci. (1990) 97: 239; Bestal, D. J. J. Cel l. Bi ol (1992) 119: 451; Mahoney, P.A. et al. Cell. (1991) 853; Dantzig, A.H. et al. Sci ence (1994) 264: 430; and Sano, K. et al. EMBO J. (1993) 12: 2249. The inclusion of the BT-R? in the superfamily it is further supported by the report that EDTA decreases when the BT CrylAB toxin is bound to the 210 kD receptor of M. sexta (Martinez-Ramirez, AC et al., Bi och em Bi ophys Res Commun (1994) 201: 782 ).

It is noted at once that the amino acid sequence of BT-R? reveals that the standard that binds calcium is present. This is consistent with the possibility that cells that have receptors to bind toxins can survive even though they develop tissues where solute-permeable ones are included and thus affect tissue disintegration. This mechanism is proposed for the death of the insects that ingest the toxin by means of the epithelial cells in their middle part of the digestive tract by Knowles, B.H. et al. Bi och em Acta (1987) 924: 509. A mechanism supported in part by the results set forth in Example 4 below is also reported which indicates that the effect of the toxin on modified 293 embryo cells to mark the receptor on its surface is reversible.

E. rDNA Molecules Containing a Nucleic Acid Molecule that Encodes BT-Ri The present invention also provides the recombinant DNA molecules (DNA) that contain the sequences encoding a BT-R? described herein, or fragments thereof, such as a soluble fragment of BT-R? which contains the toxin-BT binding site. As used herein, a rDNA molecule is a DNA molecule that has been subjected to molecular manipulation in vi tro. Methods for generating rDNA molecules are well known in the art, for example, see Sambrook et al., Mol ecu ar Cloning (1989). In the preferred rDNA molecules of the present invention, a DNA sequence encoding the BT-R? that encodes a BT-R protein? or a fragment of BT-R ?, is operably linked to one or more tag control sequences and / or sequence vectors.

The choice of vector and / or label control sequences wherein the sequence encoding the BT-R? operably linked depends directly, as is well known in the art, on the desired functional properties, for example, the labeling of the protein and the host cell to be transformed. A vector contemplated by the present invention is at least capable of directing the duplication or insertion within the host chromosome, and also preferably the labeling, of the sequence encoding the BT-R? included in the rDNA molecule.

The labeling control elements that are used to regulate the labeling of an operably linked protein encoding the sequence are known in the art and include, but are not limited to, inducible promoters, constituent promoters, secretory signals, enhancers, terminators of the transcription and other regulatory elements. Preferably, an easily controllable, inducible promoter that reacts to a nutrient in the host cell medium is used. In addition, the use of secretion signals to direct the secretion of BT-R ?, protein or fragment, out of the cell may be desirable for soluble fragments.

In one embodiment, the vector containing a nucleic acid molecule encoding the BT-Rx will include a prokaryotic duplication, for example, a DNA sequence that has the ability to direct autonomous duplication and maintenance of the recombinant DNA molecule intrachromosomally in a prokaryotic host cell, such as a bacterial host cell, transformed therewith. These duplications are well known in art. In addition, vectors that include a prokaryotic replica can also include a gene whose label confers a detectable label such as drug resistance. The typical bacterial genes resistant to the drug are those that confer resistance to ampicillin or tetracycline.

Vectors that include a prokaryotic duplication may include a prokaryotic or viral promoter capable of detecting the labeling (transcription and translation) of the sequence encoding the BT-R? in a bacterial host cell, such as E. col i. A promoter is a tag control element formed by a DNA sequence that allows the binding of RNA polymerase and for transcription to occur. Promoter sequences compatible with bacterial hosts are typically provided with plasmid vectors containing the convenient restriction sites for the insertion of a DNA segment of the present invention. Typical plasmid vectors are pUC8, pUC9, pBR322 and pBR329 available from Biorad Laboratories (Richmond, CA), pPL and pKK223 available from Pharmacia, Piscataway, NJ.

Labeling vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can also be used for variant rDNA molecules that contain a sequence encoding BT-R ?. Eukaryotic cell labeling vectors are well known in the art and are available from various commercial sources. Typically, these vectors containing convenient restriction sites are provided for the insertion of a DNA segment. Typical vectors are PSVL and pKSV-10 (pharmacia), pBPV-l / pML2d (International Biotechnologies, Inc.), pTDTl (ATCC, # 31255), the pCDMd vector described herein, and similar eukaryotic labeling vectors.

The eukaryotic cell labeling vectors used to construct the rDNA molecules of the present invention may further include a selectable marker that is effective in a eukaryotic cell, preferably a drug resistant selection marker. A preferred drug resistant marker is the gene whose label results in resistance to neomycin, for example, (neo) neomycin phosphotransferase gene. Southern et al., J. Mol. Ana l. Gene t. (1982) 1: 327-341. Alternatively, the selectable marker can be presented in a separate plasmid, and the two vectors are introduced cotransfection of the host cell, and selected by culture in the presence of an appropriate medicament for the selectable marker.

F. Host Cells Containing an Exogenously Supplied Nucleic Acid Molecule that Encodes the BT-R? .

The present invention also provides host cells transformed with a nucleic acid molecule encoding a BT-R protein? of the present invention, all the BT-R protein? or a fragment of this. The host cell can be prokaryotic or eukaryotic. Eukaryotic cells useful for the labeling of a protein BT-R? they are not limited, as long as the cell line is compatible with cell culture methods and compatible with the propagation of the vector tag and the labeling of a BT-R ?. Preferred eukaryotic host cells include, but are not limited to yeast, insect and mammalian cells, most preferred are cells that do not naturally mark a BT-R protein ?.

Any prokaryotic host can be used to label a rDNA molecule that encodes the BT-R ?. The preferred prokaryotic host is E. coli The transformation of the appropriate cellular hosts with a DNA molecule of the present invention is carried out by well-known methods that typically depend on the type of vector used and the host system employed. With respect to the transformation of prokaryotic host cells, electroporation and salt treatment methods are typically employed, see, for example, Cohen et al., Proc t Aca d. Sci. USA (1972) 69: 2110; and Maniatis et al .; Molecular Cloning, A. Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1982). With respect to the transformation of vertebrate cells with rDNA containing vectors, electroporation is typically employed and methods of treating salts or cationic lipids, see, for example, Graham et al; Virol (1973) 52: 456; Wigler et al., Proc t Na ti Aca d. Sci. USA (1979) 76: 1373-76.

Successfully transformed cells, for example, cells containing a rDNA molecule of the present invention, can be identified by well known techniques. For example, the cells resulting from the introduction of a rDNA of the present invention can be cloned to produce simple colonies.

Cells from these colonies can be cultured, used and their DNA content examined by the presence of rDNA using a method as described by Southern J. Mol. Bi ol (1975) 98: 503, or Berent et al., Bi or tech (1985) 3: 208 or the proteins produced from the cells tested by means of an immunological method.

G. The Production of a BT-R Protein? Using a rDNA Molecule The present invention also provides the methods for producing a BT-R protein? that uses one of the nucleic acid molecules that codes for BT-R? described. In general terms, the production of a BT-R? Recombinant typically involves the following steps.

First, we obtain a nucleic acid molecule that encodes a BT-R protein? or a fragment thereof, such as the nucleic acid molecule shown in Figure 1. The nucleic acid molecule, which encodes the BT-R? then they are placed in operable linkage with suitable control sequences, as described above, to generate a tagging unit containing the sequence encoding the BT-R ?. The labeling unit is used to transform a suitable host and the transformed host is cultured under conditions that allow the production of BT-R ?. Optionally the BT-R protein? was isolated from the medium or from the cells; Recovery and purification of the protein may not be necessary in some instances where some impurities can be tolerated.

Each of the above steps can be done in a variety of ways. For example, the desired coding sequences can be obtained from the genomic fragments and used directly in an appropriate host. The construction of the tag vectors that are operable in a variety of hosts is performed using an appropriate combination of duplication and control sequences. Control sequences, labeling vectors, and transformation methods are dependent on the type of host cell used to label the gene and are discussed in detail below. Suitable restriction sites can, if not normally available, be added at the ends of the coding sequences in order to provide a gene that can be removed to be inserted into these vectors. A person skilled in the art can easily adapt any host / marker system known in the art for use with the sequences encoded by the BT-R? to produce a BT-R protein ?.

- E. Identification of Cellular Agents and Constituents that bind to a BT-R Protein ?.

Another embodiment of the present invention provides methods for identifying cellular agents and constituents that bind to BT-R ?. Specifically, the cellular agents and constituents that bind to BT-R? can be identified by: 1) the ability of the agent / constituent to bind to BT-R ?, 2) the ability to block the BT-toxin that binds to BT-R ?, and / or 3) the ability to kill the cells that mark BT-R ?. The activity test for the activity BT-R? and unite and competitive assays that use a BT-R protein? are suitable for use in high performance projection methods, particularly using a soluble fragment of BT-R? containing the BT-toxin that binds the field, such as what is discussed in Example 5.

In detail, in one modality, the BT-R? it is mixed with an agent or cell extract. After mixing under conditions that allow the association of BT-R? With the agent or component of the extract, the mixture is analyzed to determine if the agent / component binds to the BT-R ?. The unions of the agents / components are identified that are capable of binding to the BT-R. Alternatively or consequently, the BT-R activity can be directly evaluated? as a means to identify agonists and antagonists of BT-R activity ?.

Alternatively, the targets that are bound by means of a BT-R protein? they can be identified using a fermentation system with two hybrids or using an assay to capture the link. In the two-hybrid fermentation system, a labeling unit that encodes a fusion protein constructs a subunit of a transcription factor with two subunits and introduces the protein BT-R? and it is marked in a yeast cell. The cell is further modified to contain 1) a labeling unit encoding a detectable label whose labeling requires the transcription factor with two subunits for labeling and 2) a labeling unit encoding a fusion protein that builds the second subunit of the label. transcription factor and a cloned segment of DNA. If the cloned segment of DNA encodes a protein that binds to the BT-R protein, the labeling causes the interaction of the BT-R? and the encoded protein. This produces that the two subunits of the transcription factor that join in the vicinity, allow the reconstitution of the transcription factor. This results in the marking of the detectable marker. The two-hybrid fermentation system is particularly useful for projecting a cDNA pattern that encodes the segments for the cell attachment of the BT-R ?.

The BT-R protein? used in the previous test can be: an isolated and fully characterized protein, a fragment of a protein BT-R? (such as a soluble fragment containing the BT-toxin that binds to the site), a cell that has been altered to label a protein / fragment of BT-R? or a fraction of a cell that has been altered to label a protein / BT-R fragment ?. In addition, the BT-R protein? can it be all the BT-R protein? or a defined fragment of the BT-R protein ?. It will be apparent to a person without experience in the art that the present assay can be used provided that the protein or fragment of BT-R? can be assayed to bind the agent, for example, by the change in molecular weight or activity.

The method is used to identify if an agent / cell component binds with a BT-R protein? Will it mainly be based on the nature of the BT-R protein? used. For example, a gel delay assay can be used to determine whether the agent binds to the BT-R? or a fragment of this. Alternatively, immunodetection and bioplate technologies can be adopted for use with the BT-R ?. A person skilled in the art can easily employ various techniques known in the art to determine if a particular agent binds to a BT-R protein ?.

Cellular agents and components can also, or alternatively, be tested for their ability to block the binding of a BT-toxin with a BT-R protein / fragment. Alternatively, the BT-toxin antibodies that bind at the site to other agents that bind to the BT-toxin that binds to the site on the BT-R protein? they can be used instead of the BT-toxin.

Cellular agents and components can be tested for the ability to modulate the activity of a BT-R protein? using a cell-free assay system or a cellular assay system. While the activities of the protein BT-R? become more defined, functional assays based on the identified activity can be employed.

As used herein, an agent is said to antagonize BT-R activity? when the agent reduces the activity of BT-R ?. The preferred antagonist will selectively antagonize BT-R ?, not affecting any other cellular protein. In addition, the preferred antagonist will reduce the activity of BT-R? in more than 50%, more preferably greater than 90%, and more preferably eliminate all BT-R activity ?.

As used herein, an agent is said to agonize BT-R activity? when the agent increases the activity of BT-R ?. The preferred agonist will selectively agonize BT-R ?, not affecting any other cellular protein. In addition, the preferred antagonist will increase the activity of BT-R? in more than 50%, more preferably greater than 90%, and more preferably more than twice the BT-R activity ?.

The agents that are tested in the above method can be randomly selected or rationally selected or designated. As used herein, an agent is said to be randomly selected when the agent is randomly chosen without considering the specific sequences of the BT-R protein? or the BT-toxin. An example of fortuitously selected agents is the use of a chemical pattern or a combinatorial pattern of peptides, or a culture broth of an organism or plant extract.

As used herein, an agent is said to be selected or rationally designated when the agent is chosen on a non-fortuitous basis that takes into account the sequence of the target site and / or its conformation in connection with the action of the agent. The agents can be rationally selected or rationally designated by using the peptide sequences that build the BT-R protein? and the BT-toxin. For example, a peptide agent selected rationally may be a peptide whose sequence is identical to the fragment of a protein BT-R? or toxin-BT.

The agents tested in the methods of the present invention may be, as examples, peptides, small molecules, and vitamin derivatives, as well as carbohydrates. A person skilled in the art will readily recognize that there are no limits to the structural nature of the agents used in the present invention in the current projection method. One class of agents of the present invention are peptide agents whose amino acid sequences that are chosen are based on the amino acid sequence of the BT-R protein? or toxin-BT. Small peptide agents can serve as competitive inhibitors of BT-R ?.

Peptide agents can be prepared using standard solid phase peptide synthesis methods (or the solution phase), as is known in the art. In addition, the DNA encoding these peptides can be synthesized using commercially available oligonucleotide synthesis instrumentation and is produced recombinantly using standard recombinant production systems. Production that uses peptide synthesis in solid phase is necessary if amino acids not encoded by a gene are included.

Another class of agents of the present invention are immunoreactive antibodies with critical compositions of the BT-R ?. As described above, the antibodies are obtained by immunization of the appropriate mammalian subjects with peptides, which contain as antigenic regions, these portions of the protein BT-R? it is projected to be labeled by antibodies. Particularly critical regions include BT-toxins that bind the field identified in Example 5. These agents can be used in competitive link studies to identify the second generation of BT-R? that unite the agents.

The cell extracts tested in the method of the present invention can be, as examples, aqueous extracts of cells or tissues, organic extracts of cells or tissues or partially purified cell fractions. A person skilled in the art will readily recognize that it is not limited to the sources of cellular extracts used in the projection method of the present invention. The preferred source for isolating BT-R cell link pairs? are cells that mark BT-R? or cells that are in close proximity to the BT-R? A profile of the projection method is as follows. The cells are modified by transfection, retroviral infection, electroporation and by other known means, to express a protein BT-R? and then it is grown under conditions where the protein is produced and projected. If desired, the cells are then recovered from the cell culture for use in the assay, or the same culture can be used per se.

In the assays, the modified cells are contacted with the candidate toxin and the effect on the metabolism or morphology in the presence and absence of the candidate is noted. The effect may be cytotoxic - for example, the cells may themselves exhibit one of the cell death rates, such as reducing thymidine output, slight increase in optical density of the culture, reduced exclusion of vital deaths (eg , trypan blue), the release of viable markers such as chromium and rubidium, and the like is increased. The differential responses between the toxin-treated cells and the absent toxin cells are then annotated. The intensity of the toxin can be evaluated by noting the intensity of the response.

These assays can be performed directly as described above or competitively with known toxins. For example, one embodiment can be made by measuring the decrease in assessing the binding of the BT toxin in the presence and absence of the candidate toxin.

In addition to simple projection candidates, the projection can be used to show improved forms of toxins which are more specific or less specific to particular kinds of insects as desired. The ability to determine binding affinity (Ka and K), dissociation and association rates and the cytotoxic effects of a candidate that allow rapid, safe and reproducible projection techniques for a large number of toxins and other ligands under conditions identical, which is not possible until now. This information will facilitate the selection of the most effective toxins and ligands for any given receptor obtained from any desired host cell.

Competition assays can also employ antibodies that are specifically immunoreactive with the receptor. These antibodies can be prepared in a conventional manner by administering the purified receptor to a vertebrate animal, monitoring the validation antibodies and recovering the antiserum or the cells that produce the antibody for immortalization, to obtain immortalized cells capable of projecting antibodies of appropriate specification. The techniques for obtaining immortalized B cells and for projecting these by secretion of the desired antibody are now conventional in the art. The resulting monoclonal antibodies may be more effective than the polyclonal antiserum as competitive reagents; In addition, the availability of the immortalized cell line that projects the desired antibodies ensures the uniformity of the production of the same reagent over time. The information and structural characteristics of the toxins and ligands tested will allow a rational method to designate more efficient toxins and ligands. Additionally, these assays will provide a better understanding of the function and structure / function relationship of the toxin / ligand and BT-R ?. analogues. In turn, this will allow the development of highly effective toxins / ligands. Ligands include natural and modified toxins, antibodies (anti-receptor and anti-ideotypic) antibodies that copy a portion of a toxin that binds to a receptor, and any of the small molecules that bind the receptors.

I. Uses of Agents that Bind to a BT-R Protein.

As provided in the background section, the BT-R? it is the target of the insecticidal activity of BT-toxins. The agents that bind a BT-R protein? can be used: 1) to kill the BT-R ?, marking cells, 2) to identify agents that block the interaction of a BT-R toxin with BT-R? and 3) in methods to identify cells that mark BT-R ?.

The methods used to use BT-R binding agents? Will they be b primarily on the nature of the binding agent with BT-R? and its intended use. For example, a BT-R? it can be used to: relea conjugated toxin into a BT-R? marker cell, modulate BT-R activity ?; directly kill the BT-R dial cells ?; or project and identify competitive union agents. An agent that inhibits the activity of BT-R? can be used to directly inhibit the growth of BT- "Rx labeling cells In addition, the identified cellular factors that bind BT-R? can, by themselves, in binding / competitive assays to identify agonists and antagonists of BT-R ?.

J. Methods to Identify the Presence of a Protein or BT-R Gene ?.

The present invention also provides methods for identifying cells, tissues or organisms by labeling a BT-R protein? or a BT-R gene ?. Each method can be used to diagnose the presence of cells or an organism that marks a BT-R protein? in vi o vi vi. The methods of the present invention are particularly useful for determining the presence of cells that are a target for BT-toxin activity or for susceptibly identifying an organism with a BT-toxin or similar agents with BT-toxin.

Specifically, the presence of a BT-R protein? it can be identified by determining whether a BT-R ?, protein or nucleic acid encoding a BT-R ?, protein is labeled in a cell, tissue or organism.

A variety of immunological and molecular genetic techniques can be used to determine if a protein BT-R? it is marked / produced in a particular cell or sample. In general, an extract containing nucleic acid molecules or an extract containing proteins is prepared. The extract is then tested to determine whether a BT-R ?, protein or a nucleic acid molecule encoding the BT-R ?, is produced in the cell.

For example, to develop a diagnostic test b on nucleic acid molecules, a suitable sample of nucleic acid is obtained and prepared using conventional techniques. DNA can be prepared, for example, simply by boiling a sample in SDS. The extracted nucleic acid can then be subjected to amplification, for example by using the polymerchain reaction (PCR) according to standard procedures, such as an RT-PCR method, to selectively amplify a nucleic acid molecule encoding BT -R? or fragment of this. The size or presence of an amplified specific fragment (typically following restriction in endonucledigestion) and then determined using gel electrophoresis or the nucleotide sequence of the fragment is determined (for example, see Webber and May Am. Gen et. (1989) 44: 388-339; Davies, J. Et al. Na ture (1994) 371: 130-136). The resulting size of the fragment or sequence is then compared to the sequences encoding the BT-R ?, proteins, for example by means of a hybridization test. Using this method, the presence of a BT-protein can be identified To develop a protein-b diagnostic test, an adequate sample of protein is obtained and prepared using conventional techniques. Protein samples can be prepared, for example, simply by mixing a sample with SDS followed by precipitation of the salt of a protein fraction. The extracted protein can then be analyzed for the presence of a BT-R protein? using known methods. For example, the presence of variants of a protein of specific size or charged using mobility in an electric field can be identified. Alternatively, the antibodies should be used for detection purposes. A person skilled in the art can easily adapt the known analytical methods of the protein to determine whether a sample contains a BT-R ?.

Alternatively, the labeling of the BT-R protein? or gene can also be used in methods to identify agents that lower the level of labeling of a BT-R gene ?. For example, the cells or tissues that mark a 'BT-R protein? may be in contact with a test agent to determine the effects of the agent on the labeling of the BT-R protein / gene ?. Agents that activate the labeling of the BT-R protein / gene can be used? as an agonist of BT-R activity? whereas agents that decrease the labeling of the BT-R protein / gene? they can be used as an antagonist of BT-R activity ?.

K. Method to sensitize the cells.

The present invention also provides methods for sensitizing cells such that they become susceptible to death with a BT-toxin, or a BT-toxin analogue. Specifically, the host cells transformed to label the BT-Rl7 receptor or a homologue of the BT-Rx receptor, becoming sensitive to the mode of action of the BT-toxins. The binding of a BT-toxin with a BT-R receptor? marked on the surface of the transformed cells causes the induction of a cell death and apoptosis of the cell that marks the receptor BT-R ?. Therefore the receiver BT-R? it is an appropriate candidate to be used in the transformation of cells where it is desired to induce cell death. There are numerous situations in which it is desired to introduce the selected gene into a selected population of cells, thus causing the death of the cell. One of these examples is in the therapeutic treatment of cancer cells. By specifically using the vectors for the delivery of the DNA molecules that encode BT-R? Within a tumor cell, tumor cells within a patient can be arranged to label a BT-R protein ?. These cells become susceptible to death with a treatment with a BT-toxin. Since BT-toxin is normally non-toxic in mammalian cells, this method is particularly applicable to induce cell death in particular cells in a mammalian host. Other situations where it may be desirable to stimulate cell death in particular cells or the progeny of the cells are in the treatment of autoimmune diseases and in the treatment of cells harboring pathogens, such as malaria or HIV agents.

The 'choice of the current steps used to introduce a DNA molecule that encodes the BT-R? Within a cell to provide cells susceptible to BT-toxin treatment is based primarily on the type of cell that is altered, the conditions under which the cell type will be altered, and all imaginable uses. A person skilled in the art can easily adapt the methods known in the art for use with the DNA molecule encoding the BT-R? of the present invention.

L. Animal Models and Gene Therapy The BT-R gene? and the BT-R protein? they can also serve as a target to generate transgenic organisms where the pattern of BT-R? It has been altered. For example, in an application, insects deficient in BT-R? or insect cells can be generated using standard successful procedures to inactivate a BT-R ?, gene, or if these animals are not viable, can antisensing molecules BT-R be used? inducible to regulate activity / marking. Alternatively, cells or an organism can be altered in order to contain a nucleic acid molecule encoding BT-R? of Manduca sexta or an antisensibilizer-BT-R dialing unit? to focus the labeling of a BT-R protein? or an antisensitizing molecule in a tissue specific form. These uses, cells or an organism, for example cells of insects or insects, are generated where the labeling of a BT-R gene? it is altered by inactivation or activation and / or replacement by a Mandu ca sex ta BT-R ?. This can be done using a variety of methods known in the art such as recombination of targets. Once the BT-Rx labeling of cells or altered organisms is generated, it can be used to 1) identify pathological and biological processes mediated by the BT-R protein?, 2) identify proteins and other genes that interact with the BT-R protein ?, 3) identify agents that can be delivered exogenously to overcome the deficiency of the BT-R protein? and 4) serve as an appropriate projection to identify mutations within the BT-R gene? that increase decrease their activity.

For example, is it possible to generate transgenic insects, such as members of the order Diptero, marking the origin of Mandu ca sixth that codes for BT-R? in a specific tissue form and test the effect of over-labeling the protein in tissues and cells that do not normally contain the BT-R protein ?.

M. Use of BT-R Generation Dialing Control Elements? The present invention also provides the tag control sequences found 5 'of the BT-R gene? newly identified in a form that can be used in generated vectors of marking. Specifically, the marking control elements BT-R? such as the BT-R? promoter, which can be easily identified to be 5 'of the start codon of the ATG in the BT-R? gene, can be used to target the labeling of a DNA sequence encoding the protein that binds .e Operably. Since the dialing of BT-R? it is mostly specific tissue, the dial control elements are particularly useful to focus the marking on a transgene introduced into a specific tissue form. A person skilled in the art can easily use the promoter of the BT-R gene? and other regulatory elements for generating labeling vectors using methods known in the art.

Without further description, it is believed that a person inexperienced in the art can, use the foregoing description and the following examples of illustration, make and use the compounds of the present invention and practice the claimed methods. The following working examples specifically indicate the preferred embodiments of the present invention, and the rest of the exposition is not constructed as limiting in any way.

Example 1 Example 1 Purification and Determination of Protein BT-R Sequence? The middle part of the intestine was extracted from M. sex ta and the BT-R protein was purified. according to the method of Vadlamodi, R. K. et al. J. Bi ol. Ch em. (1993) 268: 1233, referred to above and incorporated herein by reference. It was confirmed that the electro-eluted band contained the protein BT-R? by binding to 125 I-crylAb toxin. In gel electrophoresis, the protein was bound to the toxin that had an apparent weight of approximately 210 kD under reducing and non-reducing conditions.

The BT-R? The purified electroelution was subjected to digestion with cyanogen bromide and the cyanogen bromide fragments were separated on a high resolution 17% SDS-polyacrylamide tricine gel as described by Schagger, H. et al. Anal Bi ochem. (1987) 166: 368. The separated fragments were transferred to Problott membranes (Applied Biosystems) and five bands were removed and subjected to microsequence using standard instrumentation. The amino acid sequences obtained were (SEQ ID NOS: 18-22): 1. (Met) -Leu-Asp-Tyr-Glu-Val-Pro-Glu-Phe-Gln-Ser-Ile-Thr-Ile-Arg-Val-Val-Ala-Thr-Asp-Asn-Asn-Asp-Thr- Arg-His-Val-Gly-Val-Ala; 2. (Met) -X-Glu-Thr-Tyr-Glu-Leu-Ile-Ile-His-Pro-Phe-Asn-Tyr-Ala; 3. (Met) -X-X-X-His-Gln-Leu-Pro-Leu-Ala-Gln-Asp-Ile-Lys-Asn-His; 4. (Met) -Phe / Pro-Asn / Ile-Val-Arg / Tyr-Val-Asp-Ile / Gly; 5. (Met) -Asn-Phe-Phe / His-Ser-Va 1-Asn-Arg / Asp-Glu. Example 2 Recovery of ADNC A cDNA pattern of M. sexta was constructed from the tissue of the midgut part of the intestine using the Superscript Cholee System according to the instructions of the manufacturer (Life Technologies, Inc.). Oligonucleotide degeneration tests based on the peptide sequences determined in Example 1 were constructed using the methods and embodiment described in Zhang, S. et al. Gene (1991) 105: 61. Synthetic oligonucleotides corresponding to peptides 1-3 of Example 1 were labeled with a32P using kinase polynucleotides and used as the tests described in the standard cloning manual of Maniatis, T et al. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2nd cd 1989). A clone that hybridizes the three identified tests of 40 positive clones, the hybridization of the three tests were purified with platelet with a projection of recombinant 4X105 and subcloned into pBluescript (Stratagene). This contained an insertion of 5571 bp.

The pBluescript sequence of the cDNA was formed with double branching in both directions by the dideoxy termination method with Secuanase (USB) according to the manufacturing instructions. The sequence showed an open reading form of 4584 base pairs or 1528 amino acids together with a polyadenylation signal at position 5561. The sequence obtained and the deduced amino acid sequence is shown in Figure 1.

Thus, the deduced protein has a molecular mass of 172 kD and a pl of about 4.5. the amino acid sequences of the cyanogen bromide fragments of the native receptor are perfectly coupled within the deduced amino acid sequence. The open reading structure begins with an ATG being placed on its side by the translational initiation consensus in the GAGATGG sequence for the eukaryotic mRNAs as described by Kozak, M. Nucl ei c Aci ds Res. (1987) 15: 8125.

As shown in Figure 1, the deduced amino acid sequence includes a putative signal, shown underlined, preceding the maturation of N-terms Asn-Glu-Arg-etc. Eleven repetitions (cadl-cadll) are shown in the extracellular region upstream of the membrane region, showing with dark underlining, at position 1406-1427. The end of the 11th repetition is shown with an arrowhead. The position of the five CNBR fragments are also shown under the complete sequence.

Figure 2 compares the BT-R? obtained here with other members of the cadherin family. As the known cadherin, the external field of BT-R? It is highly repetitive and contains 11 repetitions (cadl-cadll; see Figure 2 A). The other cadherines compared in Figure 2 B are mouse cadherin P (mP ECl); Dorsophi l a fa t EC18 (EC18 fat) and protocadherin (PC42 EC2), and the intestinal transporter of Manduca sexta (HPT-l-EC-1). The eleven repetitions of the cadherin standard in BT-R? (cadl-cadll) are individually aligned with a sequence of each member of the cadherin family. The conserved waste is stored. The greatest similarity of the BT-R? with cadherines is with the extracellular repeats of the mouse cadherin-P standard. Drosophilia fat suppressor tumor and protocadherins, although the homologs are not high (homology of 20-40 and percent similarity of 30-60). The conserved BT-R repeats include AXDXD, DXE, DXNDXXP, (SEQ ID NO: 24), a glutamic acid residue and two glycine residues (Figure 2B). The A / VXDXD (SEQ ID NO: 25), DXNDN (SEQ ID NO: 26) standards are the consensus sequences for linking calcium with the two regions that occur in a cadherin repeat. In all BT-R? Repeats, the DXNDN sequence is preceded by 8 to 14 hydrophobic amino acids. Similar hydrophobic sequences have also been observed in cadherines. The length of the hydrophobic lines suggests that these areas are not transmembrane regions but represent J-lamellar structures commonly present in cadherin repeats. The BT-R? contains an assumed cytoplasmic field of 110 amino acids, smaller than the cytoplasmic field of vertebrate cadherin (116 amino acids), and shows no homology with any of the cytoplasmic fields of cadherin or with the cytoplasmic field of other proteins with which they have been compared in a database of current sequences.

To confirm that the cloned sequence encoded the entire BT-R? Protein, all the mRNA was prepared from the middle part of the intestine of M. sex ta was attached to the Northern blot by hybridization with the 4.8 kb Sacl fragment. antisensitive of the cDNA clone BT-R ?. Northern blot analysis was performed by hybridizing the test sequence at 42 ° C and 50% formamide, 5 X Denhard reagent, 50 μg / ml salmon sperm DNA and 5 X SSCP. The filtrate was then washed twice with 1 X SSC + 0.1% SDS and twice with 0.15 X SSC plus 0.1% SDS at 42 ° C. Each wash was stirred for 20 minutes. The filtrate was then exposed to an X-ray film for 24 hours. The 4.8 kb test hybridized to a single 5.6 kb band.

The clone BT-R? was translated using rabbit reticulolisate and the resulting translated products were inmonoprecipitated with cultured antiserum against the native protein encoded by BT-R ?. For the conversion vi n, the plasmid pBluescript containing the cDNA of BT-R? it was linearized and transcribed with T3 polymerase (Pharmacia). The conversion was performed according to the manufacturer's instructions with nuclease treated with rabbit reticulisate (Life Technologies, Inc.). After one hour of incubation at 30 ° C, the reaction mixture was combined with an equal volume of SDS buffer or a lysed compound was formed with 50 M Tris buffer containing 1% NP40 and 250 mM NaCl (pH 8.0 ) for immunoprecipitation. The pre-immune serum was used as a control. The conversion and the immunoprecipitation products were electrophoresed on a fixed 7.5% SDS-polyacrylamide gel, treated with the Enhancer (Dupont NEN), dried and exposed to an X-ray film for 12 hours.

Two bands of the protein of approximately 172 kD and 150 kD were obtained as determined by SDS-PAGE; it was postulated that the conversion product 150 kD due to the initiation of the conversion from an internal methionine to amino acid 242. This is consistent with the observations of Kozak, M. Mol. Cel l. Bi ol. (1989) 9: 5073.

Thus the two results confirm that the entire length of the clone was obtained.

Example 3 Recombinant Production and Characteristics of BT-R Protein? The cDNA clone of BT-R? was subcloned in the ADNpc3 mammalian labeling vector (invitrogen) and the transfected construct in COS-7 cells. Membranes isolated from transfectants COS-7 were solubilized, electrophoresis was performed and the ligand was stained with the toxin 12 SI-cryIAb. The cells were cultured 60 hours after transfection, washed with phosphate-buffered brine and lysed by freezing the liquid nitrogen. Cell membranes were prepared by differential centrifugation as described by Elshourbagy, N.A. et al. J. Bi ol. Ch em. (1993) 266: 3873. The control cells were COS-7 cells transfected with ADNpc3.

Cell membranes (10 μg) on 7.5% SDS-PAGE spots were prepared on a nylon membrane and blocked with brine buffered with Tris containing 5% fat-free dry milk powder, 5% glycerol and 1% Tween-20. The nylon membrane was then incubated with 1Z 5 I cryIAb toxin (2 X 105 cpm / ml) for 2 hours with the blocking buffer, dried and exposed to an X-ray film at -70 ° C. The labeled toxin was bound to a protein 210 ± 5 kD; the 210 kD band was observed only in the tracks containing the membranes prepared with M. sex ta or the COS-7 cells transfected with BT-R? containing 4810 bp of the cDNA comprising an open reading structure.

The discrepancy between the labeled 210 kD protein and the calculated 172 kD molecular weight is due to the glycolisation of the protein; the conversion in vi tro of the cDNA clone, as described above, which does not result from glycolisation, produces the 172 kD protein. To verify this, the COS-7 protein produced was subjected to digestion with N-glycosidase-F by first denaturing the purified protein by boiling in 1% SDS for 5 minutes followed by the addition of NP-40 to a final concentration of 1% in the presence of 0.1% SDS, and then incubated and the protein was denatured in sodium phosphate buffer, pH 8.5 at 37 ° C with N-glycosidase-F for 10 hours. The controls were incubated under the same conditions without the enzyme. The digestion products were separated in 7.5% SDS-PAGE and stained with Coomassie brilliant blue. This glucosidase treatment reduced the molecular weight of the BT-R protein? from 210 to 190 kD; this indicates the N-glycolisation of some of the 16 sites of N-glycolisation in consensus in the protein. The treatment of BT-R? with O-glycosidase and neuraminidase did not alter the mobility of the protein.

In addition, the 296 embryonic cells were transfected with the BT-R? DNA clone. at ADNpc3 and incubated with the labeled toxin (0.32 nM) in the presence of concentrations that increase (0 to 10-6 M) of unlabelled toxin. The non-specific binding was measured as the radioactive binding in the presence of an unlabeled 1M toxin. A value for the constant dissociation (Kd) of 1015 pM was determined by the Scatchard analysis; this is approximately the same value as that obtained for the natural receptor as described by Vadlamudi, R. K. et al. J. Biol. Ch em. (1993) (supra).

Example 4 Physiological Effect of BT Toxin in 293 Modified Embryonic Cells Both 293 unmodified embryonic cells, and 293 cells that have been modified to produce the BT-R receptor? as described in Example 3, when groups were formed in the form of an adherent star. When the BT toxin (200 nM) was added to the serum-free medium, the groups were pooled and released from the plastic surfaces of the culture dish. This effect was also observed under known conditions of cytoxicity for the 233 cells. The aforementioned effect was observed only when the cells were cultured in the serum free medium since the toxin binds to the serum and would not be so effective under conditions where the serum is present.

However, in the presence of the antireceptor antiserum, this effect of the BT toxin is blocked. Also, when the serum is returned to a culture of the modified E293 cells which have been treated under serum-free conditions with the toxin, the cells return to their normal star-shaped grouping forms. This indicates that the effect of the toxin is reversible.

Example 5 Identification of a Fragment of BT-R? that binds to a BT Toxin To understand some of the properties of BT-R ?, searches have been undertaken to define the locus of interaction of the BT-R protein / CrylAb-protein. The length of the wild type amino acid sequence is provided in Figure 1 with a block diagram of a possible cadherin structure for the BT-R? shown in Fig. 3. In both figures the restriction digestion sites of the cDNA are provided in relation to the position in which they would interrupt the amino acid coding sequence.

A small fragment that remains between the BamHl and Sacl restriction sites of the BT-R? Wild type was cloned into the pCITE vector (Novegen). This vector contains the transcription / conversion sequences designed for use in a rabbit reticulosite lisation system (RRL). The clone was analyzed by restriction tracing and mRNA labeling (Fig. 4). The UP marks show that the uncut plasmid and the NP and XP labels show restricted digestions using Nsil and Xhol, respectively Nsil is used because it only has one restriction site that remains within the Bam-Sac fragment and is not cut at any side inside the pCITE vector. The BSP marks show the restricted digestion of the clone using BamHl and Sacl. Digestion releases the cloned fragment which is separated into approximately 700 base pairs. The RT1 and RT2 tags show the transcription of clone mRNA after linearization with Xhol. The mRNA separates into the 1350 basic pairs.

The protein has been prepared for analysis from its clone in two ways. First, an RRL conversion kit is employed to produce the protein from the mRNA transcription reaction described above. The second, the plasmid was added to a coupled RRL-based transcription and conversion (TNT) kit. The production of the protein was detected using 35S-methionine as a brand (Fig. 5). The LCR mark shows the production of the luciferase protein from mRNA in an RRL kit and the LCT mark is the luciferase protein of a plasmid containing the sequence encoding the converted luciferase in the TNT team. Both are the positive controls to demonstrate that the two conversion equipment are operational. The major bands for luciferase conversion were observed at 66 kDa. The lines marked RRx and RR2 show the labeling of the polypeptide sequence of the BT-R? converted from mRNA into the RRL equipment. The lines TT1 and TT2 are conversions of the pCITE plasmid which contains the Bam-Sac fragment of the TNT device. The four lines have a main band at 30 kDa which is the expected size of the Bam-Sac fragment with the addition of a tag of the encoded antibody called Marca-S. The S-Mark is part of the pCITE's ulticlonation site.

The ability of the clone to bind the insecticidal toxin CrylAb was then tested on the clone. The conversion of the Bam-Sac fragment polypeptide to BT-R? was performed in duplicate as described above. The only change is that the 35S-met ionin label was left out of the reaction mixture to produce the non-radiolabelled proteins. The proteins were separated by SDS-PAGE, labeled with nitrocellulose and hybridized with 125 I-labeled Cryl? B (Fig. 6). The BBMV is the BT-R? of the wild type prepared from the membrane vesicles that are in contact with the food in the middle part of the intestine (BBMV) of M. sex ta, and is used as a positive control. The RBK and TBK are control reactions RRL and TNT prepared without the current mRNA or plasmid to determine if the protein is endogenous with the equipment that binds to CrylAb. P.? and RR2 are conversions of the RRL equipment and TTl and TT2 are of the TNT equipment. A single 30-kDa band appears in each of these lines. Two are marked by arrows. These bands show that the Bam-Sac fragment of BT-R? is able to bind the insecticidal CrylAb toxin.

For a better understanding of the nature of this binding site, a group of truncation mutants of BT-R? was prepared through the use of restriction digestions. The cDNA was digested at specific sites to eliminate the long incremental portions of the C-terminus. The restriction enzymes used were Nsil, BamHl, NruI, Clal, Xhol, and Stul (Figs 1 and 3). The procedure involves the linearization of the plasmid in each of these sites and transcribing until truncation. The short A.RNm's were then converted into a RRL-labeled nitrocellulose kit and hybridized with the 125I-labeled CrylAb. The conversion of the BT-R? Wild type of the cDNA showed binding to the 172-kDa protein band, the expected size of the BT-R? wild type. Small bands that bind to Cr-ylAb are also shown although the nature of these bands has not been determined. A blank is made by preparing an RRL reaction mixture without any mRNA given in several bands lower than 66 kDa that show the type of binding of CrylAb with the reticulosites. The specification of this union has not been determined. The truncation mutants created by the restriction digesters Nsil, BamHl, NruI, Clal, Xhol, and Stul do not show any binding to CrylAb except in the region where the teculosites bind with CrylAb. These data show that the elimination of the last 100 amino acids of the BT-R? by the restriction of Nsil causes the loss of BT-R capacity? to join CrylAb. This locates the toxin binding site in the BT-R clone? and provides a soluble fragment of the receptor that can be used in the toxin and other binding studies.

A clone of a fragment of BT-R ?, called the Bam-Sac fragment, has been prepared. It was prepared using the restriction digesters BamHl and Sacl (Fig. 1) and the cloning of the resulting fragments into a vector called pCITE. The polypeptide sequence was converted and tested by binding to the insecticidal toxin CrylAb (Figure 8). The Bam-Sac fragment binds to CrylAb, providing the first revelation within the location of the CrylAb that binds to the site within the BT-R ?. This remains in the last 234 C-terminal amino acids. This evidence is also supported by a group of truncation mutants that has been prepared. The removal of the more than 100 C-terminal amino acids from the BT-R? of the wild type causes the loss of the CrylAb junction. The C-terminal end of the BT-R? is the site of the binding site with CrylAb.

Example 6 Identification of the BT-R counterpart? that binds to a BT Toxin Western markings of tissue extracts prepared from larvae of the cotton moth and corn borer were prepared and tested with labeled Cryla (Figure 7). The results show that the homologs of the BR-R? they are present in these two insects and can be easily isolated using the methods described herein.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: UNIVERITY OF WYOMING (ii) TITLE OF THE INVENTION: RECEPTOR FOR A TOXIN OF BACILLUS THURINGIENSIS (iii) SEQUENCE NUMBER: 26 (iv) CORRESPONDENCE DIRECTION: (A) ADDRESS: MORRISON & FOERSTER LLP (B) STREET: 2000 Pennsylvania Ave. N. W. (C) CITY: Washington (D) STATE: DC (E) COUNTRY: E.U.A. (F) ZIP: 20006-1812 (v) COMPUTER READING OF: (A) MIDDLE TYPE: Floppy dis (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: Pc-DOS / MS-DOS (D) PROGRAM : Patentin Relay # 1.0, version # 1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: PCT / US98 / 11868 (B) REGISTRATION DATE: 08-JUN-1998 (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATE: (A) NUMBER OF APPLICATION: US 08 / 982,129 (B) DRAFT DATE: 01-DEC-1997 (C) CLASSIFICATION: (ii) DATE OF PREVIOUS APPLICATION: '(A) APPLICATION NUMBER: US 08 / 880,042 (B) DRAFT DATE: 20- JUN-1997 (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATE: (A) APPLICATION NUMBER: PCT / US 95/13256 (B) DRAFT DATE: 10-OCT-1995 (C) CLASSIFICATION: (vii) DATE OF PREVIOUS APPLICATION: (A) APPLICATION NUMBER: US 08 / 326,117 (B) DRAFT DATE: 19-OCT-1994 (C) CLASSIFICATION: (viii) REPRESENTATIVE INFORMATION: (A) NAME: LIVNAT, SHMUEL (B) NUMBER OF REGISTRATION: 33,949 (C) REFERENCE NUMBER: 271122003750 (ix) TELECOMMUNICATION INFORAMATION: (A) TELEPHONE: (202) 887-1500 (B) TELEFAX: (202) 887-0763 (C) TELEX: 90-4030 (2) ) INFORMATION FOR SEC. ID. NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: Base pairs 5577 (B) TYPE: nucleic acid (C) COUPLING: double (D) TOPOLOGY: linear (ii) MOLECULAR TYPE: cDNA (x) CHARACTERISTICS : (A) NAME / KEY: CDS (B) LOCATION: 197 .. 780 (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 1: GACCAATCGG AGTGTGGTGA ATTTTTGGAA AATATTTTGT GCGGTTCCTT TAGTTGTGTA 60 ATATAGTACT TTAGTTACAA ATTTGGAATA ATTTGGCAGC AAAACCATCT GCAGCAACAA 120 AATCATCTGC AGCTGCGAAA TCATCTGCAG CAGCAAAAGC ATCTTCAGGA GCGAGAAAAG 180 CCCCAAATAA TGTGAG ATG GCA GTT GAC GTC CGA ATC GCT GCC TTC CTG 229 Met Wing Val Asp Val Arg lie Wing Wing Phe Leu 1 5 10 CTG GTG TTT ATA GCG CCT GCA GTT TTA GCT CAA GAG AGA TGT GGG TAT 277 Leu Val Phe lie Ala Pro Ala Val Leu Ala Gln Glu Arg Cys Gly Tyr 15. 20 25 ATG ACC GCC ATC CCA AGG CTA CCA CGA CCG GAT AAT TTG CCA GTA CTA 325 Met Thr Ala lie Pro Arg Leu Pro Arg Pro Asp Asn Leu Pro Val Leu. 30 35 40 AAT TTT GAA GGC CAG ACA TGG AGT CAG AGG CCC CTG CTC CCC GCC CCG 373 Asn Phe Glu Gly Gln Thr Trp Ser Gln Arg Pro 'Leu Leu Pro Ala Pro 45 50 55 GAG CGG GAT GAC CTG TGC ATG GAC GCC TAC CAC GTG ATA ACÁ GCC AAC 421 Glu Arg Asp Asp Leu Cys Met Asp Wing Tyr His Val lie Thr Wing Asn 60 65 70 75 CTC GGC ACG CAG GTC ATC TAC ATG GAT GAA GAG ATA GAA GAC GAA ATC 469 Leu Gly Thr Gln Val He Tyr Met Asp Glu Glu He Glu Asp Glu He 80 85 90 ACC ATC GCC ATA CTT AAT TAT AAC GCA CCA TCA ACT CCG TTC ATT GAA 517 Thr He Wing He Leu Asn Tyr Asn Gly Pro Ser Thr Pro Phe He Glu 95 100 105 CTG CCA TTT TTA TCC GGT TCG TAC AAT CTG CTG ATG CCG GTC ATC AGG 565 Leu Pro Phe Leu Ser Gly Ser Tyr Asn Leu Leu Met Pro Val He Arg 110 115 120 AGA GTT GAC AAC GGG AGT GCA TCT CAT CAT CAC GCA AGA CAG CAT TAC 613 Arg Val Asp Asn Gly Ser Ala His His His Wing Arg Gln His Tyr 125 130 135 GAG TTG CCC GGC ATG CAG CAG TAC ATG TTC AAT GTG CGC GTG GAC GGC 661 Glu Leu Pro Gly Met Gln Gln Tyr Met Phe Asn Val Arg Val Asp Giy 140 145 150 155 CAG TCG CTG GTG GCA GGC GIG TCT CTC Gv __ ATC GTC AAC ATA GAT -GAC? C9 Gln Ser Leu Val Wing Gly Val Ser Leu Wing He Val Asn He Asp Asp 160 165 170 AAC GCG CCC ATC ATA CAA AAC TTC GAG CCT TGC CGG G TT CCT GAA Í.TU -75 'Asn Ala Pro lie He Gln Asn Phe Glu Pro Cys Arg Val Pro Glu Leu 175 180 185 GGC GAG CCA GGG TTG ACÁ GAA TGC ACÁ TAC CAÁ GTA TCG GAC GCG GAC 805 Gly Glu Pro Gly Leu Thr Glu Cys Thr Tyr Gln Val Ser Asp Wing Asp 190 195 200 GGA CGG ATC AGC ACÁ GAG TTC ATG ACG TTC AGG ATC GAC AGC GTT CGT 853 Gly Arg He Ser Thr Glu Phe Met Thr Phe Arg He Asp Ser Val Arg 205 210 215 GGC GAG GAG GAG ACC TTC TAC ATC GAA CGG ACG AAT ATC CCC AAC CAA 901 Gly Asp Glu Glu Thr Phe Tyr He Glu Arg Thr Asn He Pro Asn Gln 220 225 230 235 TGG ATG TGG CTA AAT ATG ACC ATA GGC GTT AAT ACC TCG CTC AAC TTC 949 Trp Met Trp Leu Asn Met Thr He Gly Val Asn Thr Ser Leu Asn Phe 240 245 250 GTC ACC AGT CCG CTG CAT ATA TTC AGC GTG ACÁ GCC CTG GAC TCG CTC 997 Val Thr Ser Pro Leu His He Phe Ser Val Thr Ala Leu Asp Ser Leu 255 260 265 CCG AAC ACC CAC ACG GTG ACT ATG ATG GTG CAA GTG GCG AAT GTG AAC 1045 Pro Asn Thr His Thr Val Thr Met Met Val Gln Val Wing Asn Val Asn 270, 275 280 AGC CGT CCG CCG CGC TGG CTG GAG ATC TTC GCT GTC CA CA TTT GAA 1093 Ser Arg Pro Pro Arg Trp Leu Glu He Phe Wing Val Gln Gln Phe Glu 285 290 295 GAG AAA TCT TAC CAA AAC TTC ACA GTG AGG GCG ATC GAC GAC GAC ACT 1141 Glu Lys Ser Tyr Gln Asn Phe Thr Val Arg Wing He Asp Gly Asp Thr 300 305 310 315 GAG ATC AAT ATG CCT ATC AAC TAC AGG CTG ATC ACA AAT GAG GAA GAC 1189 Glu He Asn Met Pro He Asn Tyr Arg Leu He Thr Asn Glu Glu Asp 320 325 330 ACÁ TTC TTC AGC ATT GAG GCC CTG CCT GGT GGA AAA AGC GGG GCT GTA 1237 Thr Phe Phe Ser He Glu Ala Leu Pro Gly Gly Lys Ser Gly Ala Val 335 340 345 TTC CTC GTG TCG CCA ATT GAC CGC GAC ACÁ CTG CAGA CGA GAG GTG TTT 1285 Phe Leu Val Ser Pro He Asp Arg Asp Thr Leu Gln Arg Glu Val Phe 350 355 360 CCA CTT ACG ATC GTC GCT TAC AAA TAT GAT GAG GAG GCC TTC TCC ACA 1333 Pro Leu Thr He Val Wing Tyr Lys Tyr Asp Glu Glu Wing Phe Ser Thr 365 370 375 TCA ACA AAC GTG GTC ATC ATT GTG AC GAC ATC AAC GAC CAA AGA CCT 1381 Ser Thr Asn Val Val He He Val Thr Asp He Asn Asp Gln Arg Pro 380 385 390 395 GAA CCT ATA CAC AAG GAA TAT CGA r "-t- GCA ATC ATG GAG GAG ACG CCC Glu Pro He His Lys Glu Tyr Arg Leu Wing He Met Glu Glu Thr Pro 400 405 410 CTG ACC CTC AAC TTC GAT AAA GAA TTC GGA CAT GAT AAG uni TTA 1477 Leu Thr Leu Asn Phe Asp Lys Glu Phe G? And Phe His Asp Lys Asp Leu 415 420 425 GGT CAA AAC GCT CAG TAC ACG GTG CGT CTA GAG AGC GTG GAC CCT CCA 1525 Gly Gln Asn Wing Gln Tyr Thr Val Arg Leu Glu Ser Val Asp Pro Pro 430 435 440 GGC GCT GCT GAG GTC TTC TAC ATA GCG CCT GAA GTC GGC TAC CAG CGA 1573 Gly Wing Wing Glu Wing Phe Tyr He Wing Pro Glu Val Gly Tyr Gln Arg 445 450 455 CAG ACC TTC ATC ATG GGC ACC CTC AAT CAC TCC ATG CTG GAT TAC GAA 1621 Gln Thr Phe He Met Gly Thr Leu Asn His Met Met Leu Asp Tyr Glu 460 465 470 475 GTG CCA GAG TTT CAG AGT ATT ACG ATT CGG GTG GTA GCG ACC GAC AAC 1669 Val Pro Glu Phe Gln Ser He Thr He Arg Val Val Wing Thr Asp Asn 480 485 490 AAC G AC ACG AGG CAC GTG GGC GTC GCG TTG GTT CAC ATT 'GAC CTC ATC 1717 Asn Asp Thr Arg His Val Gly Val Ala Leu Val His He Asp Leu He 495 500 505 AAT TGG AAC GAT GAG CAG CCG ATC TTC GAA CAC GCC GTG CAG ACC GTC 1765 Asn Trp Asn Asp Glu Gln Pro He Phe Glu His Wing Val Gln Thr Val 510 5i5 520 ACC TTC GAC GAG ACT GAA GGC GAG GGG TTC TTC GTC GCC AAG GCG GTT 1813 Thr Phe Asp Glu Thr Glu Gly Glu Gly Phe Phe Val Ala Lys Ala Val 525 530 535 GCA CAC GAC AGA GAC ATC GGG GAT GTC GTC GAG CAT ACT TTA TTG GGT 1861 Wing His Asp Arg Asp He Gly Asp Val Val Glu His Thr Leu Leu Gly 540 545 550 555 AAC GCT GTT AAC TTC CTG ACC ATC GAC AAA CTC ACC GGC GAC ATC CGC 1909 Asn Wing Val Asn Phe Leu Thr He Asp Lys Leu Thr Gly Asp He Arg 560 565 570 GTC TCA GCT AAC GAC TCC TTC AAC TAC CAT CGA GAA AGT GAA TTA TTT 1957 Val Ser Wing Asn Asp Ser Phe Asn Tyr His Arg Glu Ser Glu Leu Phe 575 580 585 GTG CAG GTG CGA GCT ACA GAC ACG CTG GGC GAA CCC TTC CAC ACG GCG 2005 Val Gln Val Arg Ala Thr Asp Thr Leu Gly Glu Pro Phe Hi s Thr Ala 590 595 600 ACG TCA CAG CTG GTC ATA CGA CTA AAT GAC ATC AAC AAC ACAC CCA CCC 2053 Thr Ser Gln Leu Val He Arg Leu Asn Asp He Asn Asn Thr Pro Pro 605 610 615 ACC TTA CGG CTG CCT CGA GGC AGT CCC CAA GTG GAG GAG AAC GTG CCT 2101 Thr Leu Arg Leu Pro Arg Gly Ser Pro Gln Val Glu Glu Asn Val Pro 620 625 630 635 GAT GGC CAC GTC ATC ACC CAG GAG TTA CGC GCC ACC GAC CCC GAC ACC: i49 Asp Gly His Val He Thr Gln Glu Leu Arg Wing Thr Asp Pro Asp Thr 640 645 650 ACG GCC GAT CTG CGC TTC GAG ATA AAC TGG GAC ACC TCT TTC GCC ACC 2191 Thr Wing Asp Leu Arg Phe Glu He Asn Trp Asp Thr Ser Phe Ala Thr 655 660 665 AAG CAA GGC CGC CAG GCT AAC CCC GAC GAG TTT AGG AAT TGC GTG GAA 2245 Lys Gln Gly Arg Gln Wing Asn Pro Asp Glu Phe Arg Asn Cys Val Glu 670 675 680 ATC GAG ACC ATC TTC CCC GAG ATT AAC AAC CGG GGA CTG GCT ATC GGC 1293 He Glu Thr He Phe Pro Glu He Asn Asn Arg Gly Leu Wing He Gly 685 690 695 CGC GTT GTA GCG CGC GAA ATC AGA CAC AAC GTG ACC ATA GAC TAC GAG 2341 Arg Val Val Ala Arg Glu He A rg His Asn Val Thr He Asp Tyr Glu 700 705 710 715 GAG TTT GAG GTC CTC TCC CTC HERE GTG AGG GTG CGT GAC CTT AAC ACC 2389 Glu Phe Glu Val Leu Ser Leu Thr Val Arg Val Arg Asp Leu Asn Thr 720 725 730 GTC TAC GAC GAC GAC TAC GAC GAA TCG ATG CTC ACA ATA ACT ATTA ATC 2437 Val Tyr Gly Asp Asp Tyr Asp Glu Being Met Leu .Thr He Thr He He 735 740 745. GAT ATG AAC GAC AAC GCG CCG GTG TGG GTG GAG GGG ACT CTG GAG CAG 2485 Asp Met Asn Asp Asn Ala Pro Val Trp Val Glu Gly Thr Leu Glu Gln 750 755 760 AAC TTC CGA GTC CGC GAG ATG TCG GCG GGC GGG CTC GTG GTG GGC TCC 2533 Asn Phe Arg Val Arg Glu Met Be Wing Gly Gly Leu Val Val Gly Ser 765 770 775 GTG CGC GCG GAC GAC ATC GAC GGA CCG CTC TAC 'AAC CAG GTG CGA TAC 2581 Val Arg Wing Asp Asp Asp Asp Gly Pro Leu Tyr Asn Gln Val Arg Tyr 780 785 790 795 ACC ATT TTC CCT CGT GAA GAC ACÁ GAT AAG GAC CTG ATA ATG ATC GAC 2629 Thr He Phe Pro Arg Glu Asp Thr Asp Lys Asp Leu He Met Asp 800 805 810 TTC CTC ACG GGT CAA ATT TCC GTG AAC AGA GGC GCC ATC GAC GCG 2677 Phe Leu Thr Gly Gln He Ser Val Asn Thr Ser Gly Ala He Asp Wing 815 820 825 GAT ACT CCT CCA CGC TTC CAC CTC TAC TAT HERE GTG GTC GCT AGT GAC 2725 Asp Thr Pro Pro Arg Phe His Leu Tyr Tyr Thr Val Val Wing As Asp 830 835 840 CGA TGC TCG ACA GAA GAT CCT GCA GAT TGC CCC CCT GAC CCG ACT TAT 2773 Arg Cys Ser Thr Glu Asp Pro Wing Asp Cys Pro Pro Asp Pro Thr Tyr 845 850 855 TGG GAA ACC GAA GGA AAT ATC ATÁ CAC ATC ACC GAC ACG AAC AAC 2821 Trp Glu Thr Glu Gly Asn He Thr He His Thr Asp Thr Asn Asn 860 865 870 875 AAG GTC CCG CAG GCG GAA ACG ACT AAG TTC GAT ACC GTC GTG TAT ATT 2569 Lys Val Pro Gln Wing Glu Thr Thr Lys Phe Asp Thr Val Val Tyr He 880 885 890 TAC GAG AAC GCA ACC CAC TTA GAC GAG GTG GTC ACT CTG ATA GCC AGT 29: Tyr Giu Asn Wing Thr His Leu Asp Glu Val Val Thr Leu He Wing Ser 895 900 905 GAT CTT GAC AGA GAC GAA ATA TAC CAC ACG GTG AGC TAC GTC ATC AAT 29TS5 Asp Leu Asp Arg Asp Glu He Tyr His Thr Val Ser Tyr Val He Asn 910 915 920 TAT GCA GTG AAC CCT CGA CTG ATG AAC TTC TTC TCC GTG AAC CGA GAG 3013 Tyr Wing Val Asn Pro Arg Leu Met Asn Phe Phe Ser Val Asn Arg Glu 925 930 935 ACC GGC CTG GTG TAC GTG GAC TAT GAG ACC CAG GGT AGT GGC GAG GTG 3061 Thr Gly Leu Val Tyr Val Asp Tyr Glu Thr Gln Gly Ser Gly Glu Val 940 945 950 '955 CTG GAC CGT GAT GGT GAT GAA CCA ACG CAC CGT ATC TTC TTC AAC CTC 3109 Leu Asp Arg Asp Gly Asp Glu Pro Thr His Arg He Phe Ashe Leu 960 965 970 ATC GAC AAC TTC ATG GGG GAA GGA GAA GGT AAC AGA AAT CAG AAC GAC 3157 He Asp Asn Phe Met Gly Glu Gly Glu Gly Asn Arg Asn Gln Asn Asp 975 980 985 ACA GAA GTT CTC GTT ATC TTG GAT GTG AAT GAC AAT GCT CCT GAA 3205 Thr Glu Val Leu Val He Leu Leu Asp Val Asn Asp Asn Ala Pro Glu 990 995 1000 TTG CCA CCG CCG AGC GAA CTC TCT TGG ACT ATA TCT GAG AAC CTT AAG 3253 Leu Pro Pro Pro Ser Glu Leu Ser Trp Thr He Ser Glu Asn Leu Lys 1005 1010 1015 CAG GGC G CT CGT CTT GAA CCA CAT ATC TTC GCC CCG GAC CGC GAC GAG 3301 Gln Gly Val Arg Leu Glu Pro His He Phe Wing Pro Asp Arg Asp Glu 1020 1025 1030 1035 CCC GAC ACÁ GAC AAC TCC AGG GTC GGC TAC GAG ATC CTG AAC CTC AGC 3349 Pro Asp Thr. Asp Asn Ser Arg Val Gly Tyr Glu He Leu Asn Leu Ser 1040 1045 1050 ACG GAG CGG GAC ATC GAA GTG CCG GAG CTG TTT GTG ATG ATTA CAG ATC 3397 Thr Glu Arg Asp He Glu Val Pro Glu Leu Phe Val Met He Gln He 1055 1060 _ 1065 GCG AAC GTC ACG GGA GAG CTG GAG ACC GCC ATG GAC CTC AAG GGA TAT 3445 Wing Asn Val Thr Gly Glu Leu Glu Thr Wing Met Asp Leu Lys Gly Tyr 1070 1075 1080 TGG GGG ACG TAC GCT ATA CAT ATA CGG GCA TTC GAC CAC GGC ATT CCG 3493 Trp Gly Thr Tyr Wing He His He Arg Wing Phe Asp His Gly He Pro 1085 1090 1095 CAA ATG TCC ATG AAC GAG HERE TAT GAG CTG ATC ATC CAT CCG TTC AAC 3541 G? N Met Ser Met Asn Glu Thr Tyr Glir Leu He He His Pro Phe Asn 1100 '1105 1110 1115 TAC TAC GCG CCT GAG TTC GTC TTC CCG ACC AAC GAT GCC GTC ATA CGA 3589 Tyr Tyr Wing Pro Giu Phe Val Phe Pro Thr Asr. Asp Wing Val He Arg 1120 1125 1130 CTT GCG AGG GAA CGA GCT GTA ATC AAT GGA GTT CTA GCG HERE GTG AAC Leu Wing Arg Glu Arg Wing Val He Asn Gly Val Leu Aia Thr Val Asn 1135 1140 1145 GGA GAG TTC TTG GAG CGG ATA TCG GCG ACT GAT CCG GAC GGA CTC CAC 3685 Gly Glu Phe Leu Glu Arg He Ser Wing Thr Asp Pro Asp Gly Leu His 1150 1155 1160 GCG GGC GTC GTC ACC TTC CAG GTG GTA GGC GAT GAG GAA TCA CAG CGG 3733 Wing Gly Val Val Thr Phe Gln Val Val Gly Asp Glu Glu Ser Gln Arg 1165 1170 1175 TAC TTT CAA GTA GTT AAC GAT GGC GAC AAC CTC GGC TCG TTG AGG TTA 3781 Tyr Phe Gln Val Val Asn Asp Gly Glu Asn Leu Gly Ser Leu Arg Leu 1180 1185 1190 1195 CTG CA GCC GTT CCA GAG GAG ATC AGG GAG TTC CGG ATA ACG ATT CGC 3829 Leu Gln Wing Val Pro Glu Glu He Arg Glu Phe Arg He Thr He Arg 1200 1205 1210 GCT ACA GAC CAG GGA ACG GAC CCA GGA CCG CTG TCC ACG GAC ATG ACG 3877 Thr Wing Asp Gln Gly Thr Asp Pro Gly Pro Leu Ser Thr Asp Met Thr 1215 1220 1225 TTC AGA GTT GTT TTT GTG CCC ACG CAGA GGA GAA CCT AGA TTC GCG TCC 3925 Phe Arg Val Val Phe Val Pro Thr Gln Gly Glu Pro Arg Phe Wing Ser 1230 1235 1240 TCA GAA CAT GCT GTC GCT TTC ATA GAA AAG AGT GCC GGC ATG GAA GAG 3973 Ser Glu His Wing Val Wing Phe He Glu Lys Ser Wing Gly Met Glu Glu 1245 1250 -1255 TCT CAC CAÁ CTT CCT CTA GCA CA CA GAC ATC AAG CAT CAT CTC TGT GAA 4021 Ser His Gln Leu Pro Leu Ala Gln Asp He Lys Asn His Leu Cys Glu 1260 1265 1270 1270 1275 GAC GAC TGT CAC AGC ATT TAC TAT CGT ATT ATC GAT GGC AAC AGC GAA 4069 Asp Asp Cys His Ser He Tyr Tyr Arg He He Asp Gly Asn Ser Glu 1280 1285 1290 GGT CAT TTC GGC CTG GAT CCT GTT CGC AAC AGG TTG TTC CTG AAG AAA 4117 Gly His Phe Gly Leu Asp Pro Val Arg Asn Arg Leu Phe Leu Lys Lys 1295 1300 1305 GAG CTG ATA A GG GAA CAA AGT GCC TCC CAC ACT CTG CAA GTG GCG GCT 4165 Glu Leu He Arg Glu Gln Ser Ala Ser His Thr Leu Gln Val Ala Wing 1310 1315 1320 AGT AAC TCG CCC GAT GGT GGC ATT CCA CTT CCT GCT TCC ATC CTT ACT 4213 Ser Asn Ser Pro Asp Gly Gly Pro Leu Pro Wing Be He Leu Thr 1325 1330 1335 GTC ACT GTT ACC GTG AGG GAG GAC CCT CGT CCA GTG TTT GTG AGG 4261 Val Thr Val Thr Val Arg Glu Wing Asp Pro Arg Pro Val Phe Val Arg 1340 1345 1350 1355 GAA TTG TAC ACC GCA GGG ATA TCC ACA GCG GAC TCC ATC GGC AGA GAG 4309 Glu Leu Tyr Thr Wing Gly He Ser Thr Wing Asp Ser He Gly Arg Glu 1360 1365 L370 CTG CTC AGA TTA CAT GCG ACC CAG TCT GAA GGC TCG GCC ATT ACT TAT 4357 Leu Leu Arg Leu His Wing Thr Gln Ser Glu Gly Ser Wing He Thr Tyr 1375 1380 1385 GCT ATA GAC TAC GAT ACA ATG GTA GTG GAC CCC AGC CTG GAG GCA GTG 4405 Wing He Asp Tyr Asp Thr Met Val Val Asp Pro Ser Leu Glu Val Wing 1390 1395 1400 AGA CAG TCG GCT TTC GTA CTG AAC GCT CAA ACC GGA GTG CTG ACG CTT 4453 Arg Gln Ser Ala Phe Val Leu Asn Ala Gln Th r Gly Val Leu Thr Leu 1405 1410 1415 AAT ATC CAG CCC ACG GCC ACG ATG CAT GGA CTG TTC AAA TTC GAA GTC 4501 Asn He Gln Pro Thr Wing Thr Met His Gly Leu Phe Lys Phe Glu Val 1420 1425 1430 1435 ACÁ GCT ACT GAC ACG GCC GGC GCT CAG GAC CGC ACC GAC GTC ACC GTG 549 Thr Wing Thr Asp Thr Wing Gly Wing Gln Asp Arg Thr Asp Val Thr Val 1440 1445 1450 TAC GTG GTA TCC TCG CAG AAC CGC GTC TAC TTC GTG TTC GTC AAC ACG 4597 Tyr Val Val Ser Ser Gln Asn Arg Val Tyr Phe Val Phe Val Asn Thr 1455 1460 1465 CTG CAA CAG GTC GAA GAC AAC AGA GAC TTT ATC GCG GAC ACC TTC AGC 4645 Leu Gln Gln Val Glu Asp Asn Arg Asp Phe He Wing Asp Thr Phe Ser 1470 1475 1480 'GCT GGG TTC AAC ATG ACC TGC AAC ATC GAC CAG GTG GTG CCC GCT AAC' 4693 Wing Gly Phe Asn Met Thr Cys Asn He Asp Gln Val Val Pro Wing Asn 1485 1490 1495 GAC CCC GTC ACC GGC GTG GCG CTG GAG CAC AGC ACG CAG ATG GCG GCC 4741 Asp Pro Val Thr Gly Val Ala Leu Glu His Ser Thr Gln Met Ala Ala 1500 1505 1510 1515 ACT TCA TAC GGG ACÁ ACG TAC CCG TAC TCG CTG ATG A GA TAGACAGATC 4790 Tbr Ser Tyr Gly Thr Thr Tyr Pro Tyr Ser Leu Met Arg 1520 1525 CGTAGTGACC TAGTCCTCCT GAGCTCGATA CAAACAACGC TGGCGGCGCG ATCGTGGTGT 4850 TGCAGGACTT GTTGACCAAC TCCAGCCCGG ACTTCGGCGC CTGACTCGAG CCTGCACGGT 4910 GTACGTCTGG CCTCACTGTC TGCTGTGCTC GGTTTCATGT GCCTTGTGCT ACTGCTTACC 4970 TTCATCATCA GGACTAGAGC GCTAAACCGA CGGTTGGAAG CCCTGTCGAT GACGAAGTAC 5030 GGCTCACTGG ACTCTGGATT GAACCGCGCC GGCATCGCCG CCCCCGGCAC CAACAAACAC 5090 ACTGTGGAAG GCTCCAACCC TATCTTCAAT GAAGCAATAA AGACGCCAGA TTTAGATGCC 5150 ATTAGCGAGG GTTCCAACGA CTCTGATCTG ATCGGCATCG AAGATCTTGC GCACTTTGGC 5210 AACGTCTTCA TGGATCCTGA GGTGAACGAA AAGGCAAATG GTTATCCCGA AGTCGCAAAC 5270 CACAACAACA ACTTCGCTTT CAACCCGACT CCCTTCTCGC CTGAGTTCGT TAACGGACAG 5330 TTCAGAAAGA TCTAGAAGAT AACAACACTA GTTAAGATCA TTAATTTTGG AGTTTGGAAT 5390 TAAGATTTTT GAAAGGATAG TTGTGATAAG CCTGTGATTT TTAAAACTGT AATTGAAAAA 3-_3v AAAAA7TGAG ACCTCCATTT AAGCTCTTGC TCTCATCTCA TCAAATTTTA TAAAATGCCA 5510 TTAGTCATTA AGATACTCGA TTTAATTTAA GATTATTTAA GATATTATGT AAAATAAATA 5570 TATTGTC -5577 (2) INFORMATION FOR SEC. ID. NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1528 amino acids (B) TYPE: amino acid (C) TOPOLOGY: linear (ii) MOLECULAR TYPE: protein (Xi) DESCRIPTION OF SEQUENCE: SEC. ID. NO: 2: Met Ala Val Asp Val Arg He Ala Ala Phe Leu Leu Val Phe He Ala 1 5 10 15 Pro Ala Val Leu Ala Gln Glu Arg Cys Gly Tyr Met Thr Ala He Pro 20 25 30 Arg Leu Pro Arg Pro Asp Asn Leu Pro- Val Leu Asn Phe Glu Gly Gln 35 40 45 Thr Trp Ser Gln Arg Pro Leu Leu Pro Wing Pro Glu Arg Asp Asp Leu 50 55 60 Cys Met Asp Wing Tyr His Val He Thr Wing Asn Leu Gly Thr Gln Val 65 70 75 80 He Tyr Met Asp Glu Glu He Glu Asp Glu He Thr He Wing He Leu 85 90 '95 Asn Tyr Asn Gly Pro Ser Thr Pro Phe He Glu Leu Pro Phe Leu Ser 100 105 110 Gly Ser Tyr Asn Leu Leu Met Pro Val He Arg Arg Val Asp Asn Gly 115 120 125 Ser Ala Ser His His His Wing Arg Gln His Tyr Glu Leu Pro Gly Met 130 135 140 Gln Gln Tyr Met Phe Asn Val Arg Vai Asp Gly Gln Ser Leu Vái Wing 145. 150 155 160 Gly Val Ser Leu Wing He Asn Val Asn Asp Asn Asn Wing Pro He He 165 170 175 Gln Asn Phe Glu Pro Cys Arg Val Pro Glu Leu Gly Glu Pro Gly Leu 180 185 190 Thr Glu Cys Thr Tyr Gln Val Ser Asp Wing Asp Gly Arg He Ser Thr 195 200 205 Glu Phe Met Thr Phe Arg He Asp Ser Val Arg Gly Asp Glu Glu Thr 210 215 220 Phe Tyr He Glu Arg Thr Asn He Pro Asn Gln Trp Met Trp Leu Asn 225 230 235 -240 Met Thr He Gly Val Asn Thr Ser Leu Asn Phe Val Thr Ser Pro Leu 245 250 255 His He Phe Ser Val Thr Ala Leu Asp Ser Leu Pro Asn Thr His Thr 260 265 270 Val Thr Met Met Val Gln Val Wing Asn Val Asn Ser Arg Pro Pro Arg 275 280 285 Trp Leu Glu He Phe Wing Val Gln Gln Phe Glu Glu Lys Ser Tyr Gln 290 295 300 Asn Phe Thr Val Arg Ala He Asp Gly Asp Thr Glu He Asn Met Pro 305 310 315 '320 He Asn Tyr Arg Leu He Thr Asn Glu Glu Asp Thr Phe Phe Ser He 325 330 335 Glu Wing Leu Pro Gly Gly Lys Ser Gly Wing Val Phe Leu Val Ser Pro 340 345 350 He Asp Arg Asp Thr Leu Gln Arg Glu Val Phe. Pro Leu Thr He Val 355 360 365 Wing Tyr Lys Tyr Asp Glu Glu Wing Phe Ser Thr Ser Thr Asn Val Val 370 375 380 He He Val Thr Asp He Asn Asp Gln Arg Pro Glu Pro He His Lys 385 390 395"400 Glu Tyr Arg Leu Wing He Met Glu Glu Thr Pro Leu Thr Leu Asn Phe 405 410 415 Asp Lys Glu Phe Gly Phe His Asp Lys Asp Leu Gly Gln Asn Wing Gln 420 425 430 Tyr Thr Val Arg Leu Glu Ser Val Asp Pro Pro Gly Ala Wing Glu Wing 435 440 445 Phe Tyr He "Wing Pro Glu Val Gly Tyr Gln Arg Gln Thr Phe He Met 450 455 460 Gly Thr Leu Asn His Ser Met Leu Asp Tyr Glu Val Pro Glu Phe Gln 465 470 475 480 Be He Thr He Arg Val Val Wing Thr Asp Asn Asn Asp Thr Arg His 485 490 495 Val Gly Val Ala Leu 'vái His He Asp Leu He Asn Trp Asn Asp Glu 500 505 510 Gln Pro He Phe Glu His Wing Val Gln Thr Val Thr Phe Asp Glu Thr 515 520 525 Glu Gly Glu Gly Phe Phe Val Ala Lys Ala Val Ala His Asp Arg Asp 530 535 540 He Gly ASD Val Val Glu His Thr Leu Leu Gly Asn Ala Val Asn Phe 5 5 * 550 555 560 Leu Thr He Asp Lys Leu Thr Gly Asp He Arg Val Ser Wing Asn ASD 565 570 * 575 Being Phe Asn Tyr His Arg Glu Being Glu Leu Phe Val Gln Val Arg Wing 580 585 590 Thr Asp Thr Leu Gly Glu Pro Phe His Thr Wing Thr Ser Gln Leu Val 595 600 605 He Arg Leu Asn Asp He Asn Asn Thr Pro Pro Thr Leu Arg Leu Pro 610 615 620 Arg Gly Ser Pro Gln Val Glu Glu Asn Val Pro Asp Gly His Val He 625 • 630 635 640 Thr Gln Glu Leu Arg Wing Thr Asp Pro Asp Thr Thr Wing Asp Leu Arg 645 650 655 Phe Glu He Asn Trp Asp Thr Ser Phe Wing Thr Lys Gln Gly Arg Gln 660 665 670 Wing Asn Pro Asp Glu Phe Arg Asn Cys Val Glu He Glu Thr He Phe 675 _ 680 685 Pro Glu He Asn Asn Arg Gly Leu Ala He Gly Arg Val Val Ala Arg 690 695 700 Glu He Arg His Asn Val Thr He Asp Tyr Glu Glu Phe Glu Val Leu 705 710 715 '720 Ser Leu Thr Val Arg Val Arg Asp Leu Asn Thr Val Tyr Gly Asp Asp 725 730 735 Tyr Asp Glu Being Met Leu Thr He Thr He lie As Asp Met Asn Asn 740 745 750 Pro Wing Val Trp Val Glu Gly Thr Leu Glu Gln Asn Phe Arg Val Arg 755 760 765 Glu Met Ser Wing Gly Gly Leu Val Val Gly Ser Val Arg Ala Asp Asp 770 775 780 He Asp Gly Pro Leu Tyr Asn Gln Val Arg Tyr Thr He Phe Pro Arg 785 790 795 800 Glu Asp Thr Asp Lys Asp Leu He Met Asp Phe Leu Thr Gly Gln 805 810 815 He Ser Val Asn Thr Ser Gly Wing He Asp Wing Asp Thr Pro Pro Arg 820 825 830 Phe His Leu Tyr Tyr Thr Val Val Wing Ser Asp Arg Cys Ser Thr Glu 835 840 845 Asp Pro Wing Asp Cys Pro Pro Asp Pro Thr Tyr Trp Glu Thr Glu Gly 850 855 '860 11 As He Thr He His Thr Asp Thr Asn Asn Lys Val Pro Gln Wing 865 870 875 880 Glu Thr Thr Lys Phe Asp Thr Val Val Tyr He Tyr Glu Asn Wing Thr 885 890 895 His Leu Asp Glu Val Val Thr Leu He Ala Ser Asp Leu Asp Arg Asp 900 905 910 Glu He Tyr His Thr Val Ser Tyr Val He Asn Tyr Ala Val Asn Pro 915 920 925 Arg Leu Met Asn Phe Phe Ser Val Asn Arg Glu Thr Gly Leu Val Tyr 930 935 940 Val Asp Tyr Glu Thr Gln Gly Ser Gly Glu Val Leu Asp Arg Asp Gly 945 950 955 960 Asp Glu Pro Thr His Arg He Phe Phe Asn Leu He Asp Asn Phe Met 965 970 975 Gly Glu Gly Glu Gly Asn Arg Asn Gln Asn Asp Thr Glu Val Leu Val 980 985 990 He Leu Leu Asp Val Asn Asp Asn Ala Pro Pro Glu Leu Pro Pro Ser 995 1000 1005 Glu Leu Ser Trp Thr He Ser Glu Asn Leu Lys Gln Gly Val Arg "Leu 1010 1015 1020 Glu Pro His He Phe Wing Pro Asp Arg Asp Glu Pro Asp Thr Asp Asn 1025. 1030 1035 1040 Being Arg Val Gly Tyr Glu He Leu Asn Leu Being Thr Glu Arg Asp He 1045 1050 1055 Glu Val Pro Glu Leu Phe Val Met He Gln He Wing Asn Val Thr Gly 1060 1065 1070 Glu Leu Glu Thr Wing Met Asp Leu Lys Gly Tyr Trp Gly Thr Tyr Wing 1075 1080 1085 He His He Arg Wing Phe Asp His Gly He Pro Gln Met Ser Met Asn 1090 1095 1100 Glu Thr Tyr Glu Leu He He His Pro Phe Asn Tyr Tyr Ala Pro Glu 1105 1110 1115 1120 Phe Val Phe Pro Thr Asn Asp Ala Val He Arg Leu Ala Arg Glu Arg 1125 1130 1135 Ala Val He Asn Gly Val Leu Ala Thr Val Asn Gly Glu Phe Leu Glu 1140 1145 1150 Arg He Ser Wing Thr Asp Pro Asp Gly Leu His Wing Gly Val Val Thr 1155 1160 '1165 Phe Gln Val Val Gly Asp Glu Glu Ser Gln Arg Tyr Phe Gln Val Val 1170 1175 1180 Asn Asp Gly Glu Asn Leu Gly Ser Leu Arg Leu Leu Gln Ala Val Pro 12 1185 1190 1195 1200 Glu Glu He Arg Glu Phe Arg He Thr He Arg Ala Thr Asp Gln Gly 1205 1210 1215 Thr Asp Pro Gly Pro Leu Ser Thr Asp Met Thr Phe Arg Val Val Phe 1220 1225 1230 Val Pro Thr Gln Gly Glu Pro Arg Phe Wing Ser Ser Glu His Wing Val 1235 1240 1245 Wing Phe He Glu Lys Ser Wing Gly Met Glu Glu Ser His Gln Leu Pro 1250 1255 1260 Leu Ala Gln Asp He Lys Asn His Leu Cys Glu Asp Asp Cys His Ser 1265 1270 1275 1280 He Tyr Tyr Arg He He Asp Gly Asn Ser Glu Gly His Phe Gly Leu 1285 1290 1295 Asp Pro Val Arg Asn Arg Leu Phe Leu Lys Lys Glu Leu He Arg Glu 1300 1305 1310 Gln Ser Ala Ser His Thr Leu Gln Val Wing Ala Ser Asn Ser Pro Asp 1315 1320 1325 Gly Gly Pro Pro Leu Pro Wing Be He Leu Thr Val Thr Val Thr Val 1330 1335 1340"Arg Glu Wing Asp Pro Arg Pro Val Phe Val Arg Glu Leu Tyr Thr Wing • 1345 1350 1355 1360 Gly He Ser Thr Wing Asp Ser He Gly Arg Glu Leu Leu Arg Leu His 1365 1370 1375 Wing Thr Gln Ser Glu Gly Wing Wing He Thr Tyr Wing He Asp Tyr Asp 1380 1385 1390 Thr Met Val Val Asp Pro Ser Leu Glu Wing Val Arg Gln Ser Wing Phe 1395 1400 1405 Val Leu Asn Wing Gln Thr Gly Val Leu Thr Leu Asn He Gln Pro Thr 1410 1415 1420 Wing Thr Met His Gly Leu Phe Lys Phe Glu Val Thr Wing Thr Asp Thr 1425 1430 1435 1440 Wing Gly Wing Gln Asp Arg Thr Asp Val Thr Val Tyr Val Val Ser Ser 1445 1450 1455 Gln Asn Arg Val Tyr Phe Val Phe Val Asn Thr Leu Gln Gln Val Glu 1460 1465 1470 Asp Asn Arg Asp Phe He Wing Asp Thr Phe Ser Wing Gly Phe Asn Met 1475 1480 1485 Thr Cys Asn He Asp Gln Val Val Pro Wing Asn Asp Pro Val Thr Gly 1490 1495 1500 Val Wing Leu Glu His Ser Thr Gln Met Wing Wing Thr Ser Tyr Gly Thr 1505 1510. 1515 1520 13 TI "- Tyr Pro Tyr Ser Leu Met Arg 1525 (2) INFORMATION FOR SEC. ID. NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 107 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 3: Glu Trp Val Met Pro Pro He Phe Val Pro Glu Asn Gly Lys Gly Pro 1 5 10 15 Phe Pro Gln Arg Leu Asn Gln Leu Lys Ser Asn Lys Asp Arg Gly Thr 20 25 30 Lys He Phe Tyr Tyr Ser He Thr Gly Pro Gly Wing Asp Ser Pro Pro 35 40 45 Glu Gly Val Phe Thr He Glu Lys Glu Ser Gly Trp Leu Leu Leu His 50 55 60 Met Pro Leu Asp Arg Glu Lys He Val Lys Tyr Glu Leu Tyr Gly His 65 70 75 _ 80 Wing Val Ser Glu Asn Gly Wing Ser Val Glu Glu Pro Met As As Ser 85 90 95 He He Val Val Thr Asp Gln As Asp Asn Lys Pro 100 105 (2) INFORMATION FOR SEC. ID. NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 105 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. DO NOT: Glu Asp Thr Val Tyr Ser Phe Asp He Asp Glu Asn Wing Gln Arg Gly 1 5 10 15 Tyr Gln Val Gly Gln He Val Wing Arg Asp Wing Asp Leu Gly Gln Asn 20 25 30 Wing Glri Leu Ser Tyr Gly Val Val Ser Asp Trp Ala Asn Asp Val Phe 35 40 45 14 Ser Leu Asn Pro Gln Thr Gly Met Leu Thr Leu Thr Wing Arg Leu Asp 50 55 60 Tyr Glu Glu Val Gln His Tyr He Leu He Val Gln Ala Gln Asp Asn 65 70 75 80 Gly Gln Pro Ser Leu Ser Thr Thr He Thr Val Tyr Cys Asn Val Leu 85 90 95 Asp Leu Asn Asp Asn Ala Pro He Phe 100 105 (2) INFORMATION FOR SEC. ID. NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 93 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 5: Wing Pro Pro Val He Thr Leu Wing Pro Glu Asn Thr Asn He Gly 1 5 10 15 Be Leu Phe Pro He Pro Leu Wing Be Asp Arg Asp Wing Asn Glu Leu 20 25 30 Gln Val Wing Glu Asp Gln Glu Glu Lys Gln Pro Gln Leu He Val Met 35 40 45 - Gly Asn Leu Asp Arg Glu Arg Trp Asp Ser Tyr Asp Leu Thr He Lys 50 55 60 Val Gln Asp Gly Gly Ser Pro Pro Arg Ala Thr Ser Ala Leu Leu Arg 65 70 75 80 Val Thr Val Leu Asp Thr Asn Asp Asn Ala Pro Lys Phe 85 90 (2) INFORMATION FOR SEC. ID. NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 106 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO 6: He Val Thr Glu Asn He Trp Lys Ala Pro Lys Pro Val Glu Met Val 1 5 10 15 Giu Asn Ser Thr Pro His Pro He Lys He Thr Gln Val Arg Trp Asn 20 25 30 Asp Pro Gly Wing Gln Tyr Ser Leu Val Asp Lys Glu Lys Leu Pro Arg 35 40 45 Phe Pro Phe Ser He Asp Gln Glu Gly Asp He Tyr Val Thr Pro Leu 50 55 60 Asp Arg Glu Glu Lys Asp Wing Tyr Val Phe Tyr Wing Val Wing Lys Asp 65 70 75 80 Glu Tyr Gly Lys Pro Leu Ser Tyr Pro Leu Glu He His Val Lys Val 85 90 95 Lys Asp He Asn Asp Asn Pro Pro Thr Cys 100 105 (2) INFORMATION FOR SEC. ID. NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 105 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 7: He Thr Ala Asn Leu Gly Thr Gln Val He Tyr Met Asp Glu Glu He 1 5 10 15 Glu Asp Glu He Thr He Wing He Leu Asn Tyr Asn Gly Pro Ser Thr 20 25 30 Pro Phe He Glu Leu Pro Phe Leu Ser Gly Ser Tyr Asn Leu Leu Met 35 4'0 45 Pro Val He Arg Arg Val Asp Asn Gly Ser Ala Ser His His His Wing 50 55 60 Arg Gln His Tyr Glu Leu Pro Gly Met Gln Gln Tyr Met Phe Asn Val 65 70 75 80 Arg Val Asp Gly Gln Ser Leu Val Wing Gly yal Ser Leu Wing He Val 85 90 95 Asn He Asp Asp Asn Ala Pro He He 100 105 (2) INFORMATION FOR THE SEC. ID. NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 113 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 8: 16 Gln Asn Phe Glu Pro Cys Arg Val Pro Glu Leu Gly Glu Pro Gly Leu 1 5 10 15 Thr Glu Cys Thr Tyr Gln Val Ser Asp Wing Asp Gly Arg He Ser Thr 20 25 30 Glu Phe Met Thr Phe Arg He Asp Ser Val Arg Gly Asp Glu Glu Thr 35 40 45 Phe Tyr He Glu Arg Thr Asn He Pro Asn Gln Trp Met Trp Leu Asn 50 55 60 Met Thr He Gly Val Asn Thr Ser Leu Asn Phe Val Thr Ser Pro Leu 65 70 75 80 His He Phe Ser Val Thr Ala Leu Asp Ser Leu Pro Asn Thr His Thr 85 90 95 Val Thr Met Val Met Val Gln Asn Val Val Asn Ser Arg Pro Pro Arg 100 105 110 Trp (2) INFORMATION FOR SEC. ID. NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 107 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (Xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 9: Leu Glu He Phe Wing Val Gln Gln Phe Glu Glu Lys Ser Tyr Gln Asn 1 5 10 15 Phe Thr Val Arg Wing He Asp Gly Asp Thr Glu He Asn Met Pro He 20 25 30 Asn Tyr Arg Leu He Thr Asn Glu Glu Asp Thr Phe Phe Ser He Glu 35 40 45 Wing Leu Pro Gly Gly Lys Ser Gly Wing Val Phe Leu Val He Asp Arg 50 55 60 Asp Thr Leu Gln Arg Glu Val Phe Pro Leu Thr He Val Ala Tyr Lys 65 70 75 80 Tyr Asp Glu Glu Ala- Phe Ser Thr Ser Thr Asn Val Val He He Val 17 85 90 95 phr Asp He Asn Asp Gln Arg Pro Glu Pro 100 105 (2) INFORMATION FOR SEC. ID. NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 119 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 10: He His Lys Glu Tyr Arg Leu Wing He Met Glu Glu Thr Pro Leu Thr 1 5 10 15 Leu Asn Phe Asp Lys Glu Phe Gly Phe His Asp Lys Asp Leu Gly Gln 20 25. 30 Asn Wing Gln Tyr Thr Val Arg Leu Glu Ser Val Asp Pro Pro Gly Wing 35 '40 45. Wing Glu Wing Phe Tyr He Wing Pro Glu Val Gly Tyr Gln Arg Gln Thr 50 55 60 Phe He Met Gly Thr Leu Asn His Ser Met Leu Asp Tyr Glu Val Pro 65 70 75 80 Glu Phe Gln Ser He Thr He Arg Val Val Wing Thr Asp Asn Asn Asp 85 90 95 Thr Arg. His Val Gly Val Wing Leu Val Hi's He Asp Leu He Asn Trp 100 105 110 Asn Asp Glu Gln Pro He Phe 115 (2) INFORMATION FOR SEC. ID. NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 104 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 11: Glu His Wing Val Gln Thr Val Thr Phe Asp Glu Thr Glu Gly Glu Gly 1 • 5 10 15 Phe Phe Val Wing Lys Wing Val Wing His Asp Arg Asp He Gly Asp Val ' 18 20 25 30 Val Glu His Thr Leu Leu Gly Asn Wing Val Asn Phe Leu Thr He Asp 35 40 45 Lys Leu Thr Gly Asp He Arg Val Ser Wing Asn Asp Ser Phe Tyr His 50 55 60 Arg Glu Ser Glu Leu Phe Val Gln Val Arg Ala Thr Asp Thr Leu Gly 65 70 75 30 Gln Pro Phe His Thr Wing Thr Ser Gln Leu Val He Arg Leu Asn Asp 85 90 95 He Asn Asn Thr Pro Pro Thr Leu 100 (2) INFORMATION FOR SEC. ID. NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 138 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 12: Arg • Leu Pro Arg Gly Ser Pro Gln Val Glu Glu Asn Val Pro Asp Wing 1 5 10 15 His Val He Thr Gln Glu Leu Arg Wing Thr Asp Pro Asp Thr Thr Wing 20 25 30 Asp Leu Arg Phe Glu He Asn Trp Asp Thr Be Phe Wing Thr Lys Gln 35 40 45 Gly Arg Gln Wing Asn Pro Asp Glu Phe Arg Asn Cys Val Glu He Glu 50 55 60 Thr He Phe Phe Pro Glu He Asn Asn He Asn Asn Arg Gly Leu Wing 65 .70 75 80 He Gly Arg Val Val Wing Arg Glu He Arg His Asn Thr He Asp Tyr 85 90 95 Glu Glu Phe Glu Val Leu Ser Leu Thr Val Arg Val Arg Asp Leu Asn 100 105 110 Thr Val Tyr Gly Asp Asp Tyr Asp Glu Ser Met Leu Thr He Thr He 115 120 125 He Asp Met Asn Asp Asn Ala Pro Val Trp 130 135 19 (2) INFORMATION FOR SEC. ID. : (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 124 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 13: Val Glu Gly Thr Leu Glu Gln Asn Phe Arg Val Arg Glu Met Be Wing 1 5 10 15 Gly Gly Leu Val Val Gly Ser Val Arg Wing Asp Asp He Asp Gly Pro 20 25. 30 Leu Tyr Asn Gln Val Arg Tyr Thr He Phe Pro Arg Glu Asp Thr Asp 35 40 45 Lys Asp Leu He Met He Glu Leu Pro His Gly Ser Asn Phe Arg Glu 50 55 60 His Lys Arg Arg He Asp Wing Asn Thr Pro Pro Arg Phe His Leu Tyr 65 70 75 80 Tyr Thr Val Val Wing Ser Asp Arg Cys Ser Thr Glu Asp Pro Wing Asp 85 90. 95 Cys Pro Pro Asp Pro Tyr Tyr Trp Glu Thr Glu Gly Asn He Thr He 100 105 110 His He Thr Asp Thr Asn Asn Lys Val Pro Gln Ala 115 120 (2) INFORMATION FOR SEC. ID. NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 122 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 14: Glu Thr Thr Lys Phe Asp Thr Val Val Tyr He Tyr Glu Asn Wing Thr 1 5 10 15 His Leu Asp Glu Val Val Thr Leu He Ala Ser Asp Leu Asp Arg Asp 20 25 30 Glu He Tyr His Met Val Ser Tyr Val He Asn Tyr Ala Val Asn Pro 35 40 45 Arg Leu Met Asn Phe Phe Ser Val Asn Arg Glu Thr Gly Leu Val Tyr 50 55 '60 Val ASD Tyr Glu Thr Gln Gly Ser Gly Leu Asp Arg Asp Gly Asp Giu 65 70 75 80 Pro Thr His Arg He Phe Phe Asn Leu He Asp Asn Phe Met Gly Glu 85 90 95 Gly Glu Gly Asn Arg Asn Gln Asn Asp Thr Glu Val Leu Val He Leu 100 105 110 Leu Asp Val Asn Asp Asn Ala Pro Glu Leu 115 120 (2) INFORMATION FOR SEC. ID. NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 146 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 15: Pro Pro Pro Ser Glu Leu Ser Trp Thr He Ser Glu Asn Leu Lys Gln 1 5 10 15 Gly Val Arg Leu Glu Pro His He Phe Wing Pro Asp Arg Asp Glu Pro 20 25 30 Asp Thr Asp Asn Ser Arg Val Gly Tyr Glu He Leu Asn Leu Ser Thr 35 40 45 Glu Arg Asp He Glu Val Pro Glu Leu Phe Val Met He Gln He He 50 55 60 Wing Asn Val Thr Gly Tyr Glu He Leu Asn Leu Ser Thr Glu Arg Asp 65 70 75 80 He Glu Val Pro Glu Leu Phe Val Met He Gln He Ala Asn Val Thr 85 90 95 Gly Glu Leu Glu Thr Wing Met Asp Leu Lys Gly Tyr Trp Gly Thr Tyr 100 105 110 Wing He Tyr He Leu Wing Phe Asp His Gly He Pro Gln Met Ser Met 115 120 125 Asn Glu Thr Tyr Glu Leu He He His Pro Phe Asn Tyr Tyr Ala Pro 130 135 140 Glu Phe 145 21 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 120 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 16: Val Phe Pro Thr Asn Asp Ala Val He Arg Leu Ala Arg Giu Arg Ala 1 5 10 15 Val He Asn Gly Val Leu Wing Thr Val Asn Gly Glu Phe Leu Glu Arg 20 25 30 He Ser Wing Thr Asp Pro Asp Gly Leu His Wing Gly Val Val Thr Phe 35 40 45 Gln Val Gly Asp Glu Glu Ser Gln Arg Tyr Phe Gln Val Val Asp Asn 50 55 60 Asp Gly Glu Asn Leu Gly Ser Leu Arg Leu Leu Gln Ala Val Pro Glu 65 70 75 80 Glu He Arg Glu Phe Arg He Thr He Arg Ala Thr Asp Gln Gly Thr 85 '90 95 Asp Pro Gly Pro Leu Ser Thr Asp Met Thr Phe Arg Val Val Phe Val 100 105 110 Pro Thr Gln Gly Glu Pro Arg Phe 115 120 (2) INFORMATION FOR SEC. ID. NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 112 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 17: Ala Ser Ser Glu His Ala Val Ala Phe He Glu Lys Ser Ala Gly Met 1 5 10"15 Glu Glu Ser His Gln Leu Pro Leu Wing Gln Asp He Lys Asn His Leu 20 25 30 Cys Glu Asp Asp Cys His Ser He Tyr Tyr Arg He He Asp Gly Asn 35 40 45 Ser Glu Gly His Phe Gly Leu Asp Pro Val Arg Asn Arg Leu Phe Leu 50 55 60 Lys Lys Glu Leu He Arg Glu Gln Ser Ala Ser His Thr Leu Gln Val 65 70 75 80 22 Ala Ala Ser Asn Pro Pro Asp Gly Gly Pro Pro Leu Pro Ala Be He 85 90 95 Leu Thr Val Thr Val Thr Val Arg Glu Ala Asp Pro Arg Pro Val Phe 100 105 110 (2) INFORMATION FOR SEC. ID. NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 30 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 18: Met Leu Asp Tyr Glu Val Pro Glu Phe Gln Ser He Thr He Arg Val 1 5 10, 15 Val Ala Thr Asp Asn Asn Asp Thr Arg His Val Gly Val Ala "20 25 30 (2) INFORMATION FOR SEC. ID. NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 19: Met Xaa Glu Thr Tyr Glu Leu He He His Pro Phe Asn Tyr Tyr Ala 1 5 10 15 (2) INFORMATION FOR SEC. ID. NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 20: Met Xaa Xaa Xaa His Gln Leu Pro Leu Ala Gln Asp He Lys Asn His- 23 (2) INFORMATION FOR SEC. ID. NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (ix) CHARACTERISTICS: (A) NAME / KEY: site -Modified (B) LOCATION: 2 (C) OTHER INFORMATION: / note = "This position is Phe / Pro" (ix) FEATURE: (A) NAME / KEY: Site-Modified (B) LOCATION: 3 (C) OTHER INFORMATION: / note = "This position is Asn / Ile" (ix) FEATURE: (A) NAME / KEY: Site-Modified (B) LOCATION: 5 (C) OTHER INFORMATION: / note = 'This position is Arg / Tyr "(ix) FEATURE: (A) NAME / KEY: Site-Modified (B) LOCATION: 8 (C) OTHER INFORMATION: / note =" This position is Ile / Gly "(xi) DESCRIPTION OF THE SEQUENCE: SEC. NO: 21: Met Xaa Xaa Val Xaa Val Asp Xaa 1 5 (2) INFORMATION FOR SEC. ID. NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (ix) CHARACTERISTICS: (A) NAME / KEY: site -Modified (B) LOCATION: 4 (C) OTHER INFORMATION: / note = "This position is Phe / His" (ix) FEATURE: (A) NAME / KEY: Site-Modified (B) LOCATION: 8 (C) OTHER INFORMATION: / note = "This position is Arg / Asp" (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 22: Met Asn Phe Xaa Ser Val Xaa Glu 1 5 (2) INFORMATION FOR SEC. ID. NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 23: Ala Xaa Asp Xaa Asp 1 5 INFORMATION FOR SEC. ID. NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 24: Asp Xaa Asn Asp Xaa Xaa Pro 1 5 (2) INFORMATION FOR SEC. ID. NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (ix) CHARACTERISTICS: (A) NAME / KEY: site -Modified (B) LOCATION: 1 (C) OTHER INFORMATION: / note = "Alanina (A) Valina (V)" (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 25: Xaa Xaa Asp Xaa Asp 1 5 (2) INFORMATION FOR SEC. ID. NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) COUPLING: simple (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID. NO: 26: Asp Xaa Asn Asp Asn 1 5 It is noted that in relation to this date, the best method known to the applicant to carry out the invention is the one that is clear from the present invention.

Having described the invention as above, the content of the following is claimed as property.

Claims (15)

Claims

1. A method for identifying the agents that bind to the BT-toxin receptor, characterized in that the method comprises the steps of: i) contacting an agent with a receptor that binds a BT-toxin selected from the group consisting of a) a cell that has been altered to contain a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO. : 2, b) a cell that has been altered to contain a nucleic acid molecule encoding a fragment of the amino acid sequence of SEQ ID NO: 2 that binds to a BT toxin, c) a cell that has been altered to contain a nucleic acid molecule encoding a BT-toxin receptor that hybridizes to the nucleic acid sequence of SEQ ID NO. : 1 under high restriction, d) a cell that has been altered to contain a nucleic acid molecule encoding a fragment of a BT-toxin receptor that hybridizes to the nucleic acid sequence of SEQ ID NO. : 1 under high restriction, and which binds to a BT toxin, e) an isolated protein with an amino acid sequence of SEQ ID NO: 10 2, f) an isolated fragment of a protein with an amino acid sequence of SEQ ID NO: 2, the fragment contains a BT-toxin binding domain, g) a receptor of the 15 isolated BT toxin that is encoded by a nucleic acid molecule that hybridizes to the nucleic acid sequence of SEQ ID NO: 1 under high restriction and h) an isolated fragment 20 of the BT toxin receptor which is encoded by a nucleic acid molecule that hybridizes to the nucleic acid sequence of SEQ ID NO: 1 under high restriction, and ii) determines whether the agent binds to the toxin receptor. BT.

2. The method of claim 1, characterized in that the method further comprises the step of determining whether the agent blocks the binding of a BT-toxin to the BT-toxin receptor.

3. The method of claim 1, characterized in that the cell that has been altered is a eukaryotic cell.

4. The method of claim 3, characterized in that the eukaryotic cell is an insect cell.

5. A method for identifying agents that block the binding of a BT-toxin with a BT-toxin receptor, characterized in that the method comprises the steps of: i) contacting an agent in the presence and absence of a BT-toxin, with a receptor that binds the BT-toxin selected from the group consisting of a) a cell that has been altered to contain a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 2, b) a cell that has been altered to contain 10 a nucleic acid molecule encoding a fragment of the amino acid sequence of SEQ ID NO. : 2 that binds to a BT toxin, c) a cell that has been altered to contain a 15 nucleic acid molecule encoding a BT-toxin receptor that hybridizes to the nucleic acid sequence of SEQ ID NO: 1 under high restriction, d) a cell that has been 20 altered to contain a nucleic acid molecule encoding a fragment of a BT-toxin receptor that hybridizes to the nucleic acid sequence of SEQ ID NO: 1 under high Restriction, and that binds to a BT toxin, e) an isolated protein with an amino acid sequence of SEQ ID NO: 2, f) an isolated fragment of a protein with a sequence of fragment contains a BT-toxin binding domain, g) an isolated BT toxin receptor that is encoded by a nucleic acid molecule that 10 hybridizes to the nucleic acid sequence of SEQ ID NO: 1 under high restriction and h) a fragment isolated from the BT toxin receptor that is encoded by an acid molecule Nucleic acid that hybridizes to the nucleic acid sequence of SEQ ID NO: 1 under high restriction, and ii) determine if the agent blocks the binding of the BT-toxin with the receptor 20 of the toxin-BT.

6. The method of claim 5, characterized in that the BT toxin is a member of the cry (l) -BT toxin family.

7. The method of claim 5, characterized in that the cell that has been altered is a eukaryotic cell.

8. The method of claim 7, characterized in that the eukaryotic cell is an insect cell.

9. An isolated antibody, characterized in that the antibody binds to a protein selected from the group consisting of a) a BT toxin receptor protein with an amino acid sequence of SEQ ID NO: 2, and b) a toxin-receiving protein. BT that is encoded by a nucleic acid molecule that hybridizes to the nucleic acid sequence of SEQ ID NO: 1 under high restriction, or a fragment of the antibody, where the antibody fragment binds to the BT-toxin.

10. The antibody of claim 9 is characterized in that the antibody binds to the BT-toxin receptor and blocks the binding of a BT-toxin to the receptor.

11. The antibody of claim 10, characterized in that the antibody binds to an epitope located within the terminal 232 c-amino acids of the BT-toxin receptor shown in SEQ ID NO: 2.

12. An isolated BT-toxin receptor protein, characterized in that it is selected from the group consisting of a) a BT toxin receptor protein with an amino acid sequence of SEQ ID NO: 2, and b) a toxin-receiving protein. BT that is encoded by a nucleic acid molecule that hybridizes to the nucleic acid sequence of SEQ ID NO: 1 under high restriction; c) a fragment of a BT-toxin receptor protein with an amino acid sequence of SEQ ID NO: 1; NO .: 2, the fragment is capable of binding to a BT-toxin, and d) a fragment of the BT-toxin receptor protein that is encoded by a nucleic acid molecule that hybridizes to the nucleic acid sequence of SEQ ID NO. NO .: 1 under high restriction, the fragment is able to bind a BT-toxin.

13. A method for producing the BT-toxin receptor protein, or a fragment thereof, the method comprises the steps of: i) culturing a cell that has been altered to contain a nucleic acid molecule encoding a BT-toxin receptor protein, the BT-toxin-binding fragment thereof, characterized in that the cell has been altered to contain a molecule of nucleic acid selected from the group consisting of a) a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 2, b) a nucleic acid molecule encoding a fragment of the amino acid sequence of SEQ ID NO .: 2 that binds to a BT toxin, c) a nucleic acid molecule that encodes a BT-toxin receptor that hybridizes to the acid sequence 10 nucleic acid of SEQ ID NO: 1 under high restriction, and d) a nucleic acid molecule encoding a fragment of a BT-toxin receptor that hybridizes the acid sequence Nucleic acid of SEQ ID NO: 1 under high restriction, and which binds to a BT toxin, under conditions wherein the nucleic acid molecule is labeled and ii) isolate the protein or fragment 20 toxin-BT receptor.

14. The method of claim 13 characterized in that the cell that has been altered is a eukaryotic cell.

15. The method of claim 14, characterized in that the eukaryotic cell is an insect cell.