JP2001512307A - Polynucleotides encoding proexendin and methods for making and using the same - Google Patents

Abstract

(57) [Summary] Exendin 4 is a biologically active peptide originally isolated from the venom of the American lizard (Gila monster). The invention includes polynucleotides encoding proexendin peptides, including exendins and novel peptides, as well as isolated or recombinant proexendin peptides. The present invention also includes antibodies that specifically recognize such peptides.

Description

Description: Polynucleotides encoding proexendin and methods for making and using the same Field of the invention The invention is in the field of molecular biology. More specifically, the present invention relates to cloned genes and their use in diagnostic, therapeutic and related applications. Background of the Invention The isolation from lizard venom of a novel peptide exhibiting 52% identity to mammalian GLP-1 prompted studies investigating the biological properties of this protein, named exendin 4 (Eng. (1992) J. Biol. Chem. 267, 7402-7405). Experiments with synthetic exendin 4 have provided evidence that this peptide shares a similar biological activity with mammalian GLP-1. Exendin 4 and truncated GLP-1 [7-36] -amide increased cAMP levels in guinea pig acinar cell preparations (Raufman et al., 1992, J. Biol. Chem. 267, 21432-21437). . And then, exendin 4 is shown to bind to the GLP-1 receptor, stimulate glucose-dependent insulin secretion, and increase both cAMP accumulation and insulin gene expression in cultured islet cell lines in vitro. (Goke et al., 1993, J. Biol. Chem. 268, 18950-19855). Full-length exendin 4 (1-39) is a potent agonist of GLP-1, but truncated exendin 4 (9-39) NH _Three Is a competitive antagonist of both exendin 4 and GLP-1 (Raufman, 1996, Regulatory Peptides 61: 1-18). Exendin's insulinotropic, GLP-1 similar properties suggest that this lizard peptide may also be useful in treating diabetic patients. Summary of the Invention A polynucleotide encoding a reptile exendin has been isolated and characterized. Surprisingly, the isolated polynucleotide encoded a proexendin pleiotropic peptide encoding a novel 47 amino acid peptide (47 amino acid ENTP) fused to the amino terminus of exendin . The first 23 amino acids of ENTP form a common signal peptide sequence. At the point of fusion between the novel peptide and exendin, there is a common dipeptidyl peptidase cleavage site. Cleavage at this site cleaves the 2-mer and exendin 4 consisting of residues 46 and 47. In addition, additional glycine residues were found at the carboxyl terminus of the putative exendin coding sequence. This additional glycine residue is probably cleaved during post-translational processing of exendin 4 and carboxyl-terminal amidation. Accordingly, the present invention provides a polynucleotide encoding proexendin. In one embodiment, the invention provides a polynucleotide encoding a lizard exendin 4 peptide and a 47 amino acid exendin N-terminal peptide (47 amino acid ENTP). In a related aspect, the invention provides a polynucleotide encoding a 47 amino acid ENTP. In yet another related aspect, the invention provides a polynucleotide encoding a 23 amino acid secretory signal peptide and a 22 amino acid ENTP. In an embodiment of the invention, the proexendin polynucleotides and fragments thereof are used for further gene cloning to identify structurally related polynucleotides and species homologs. In a related aspect of the invention, a polynucleotide encoding an antisense version of proexendin and fragments thereof are obtained and used to regulate expression of proexendin. Accordingly, the invention provides in one aspect thereof a polynucleotide that hybridizes to a polynucleotide encoding a lizard proexendin or its complement. Preferably, such polynucleotides will hybridize under moderately stringent conditions and encode a functionally equivalent gene product. In yet another aspect of the present invention, there is provided a polynucleotide that hybridizes under stringent conditions to a polynucleotide encoding a lizard proexendin or a complement thereof. In another aspect of the invention, the peptide encoded by proexendin is provided as a product of recombinant production in a host cell. In a related embodiment, a recombinant host cell that expresses proexendin cDNA, and an expression in which a polynucleotide that encodes a peptide encoded by proexendin is linked to an expression control sequence that functions in the selected host cell A construct is provided. In another aspect of the present invention, there is provided a substantially purified novel exendin N-terminal peptide and substantially purified exendin 4 (1-40). In yet another aspect, the invention provides specific antibodies to a 45 amino acid ENTP or a 22 amino acid ENTP, eg, for use in diagnosing a condition in which proexendin levels are altered. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a 492 polynucleotide sequence in which nucleotides 64-465 encode a lizard proexendin pleiotropic peptide. FIG. 2 shows the 87 amino acid sequence of the lizard proexendin gene product. Dashed lines indicate the novel ENTP sequence and the pro-exendin carboxyl-terminal region that differs from the published sequence of the exendin protein. Detailed description of the invention and preferred embodiments The invention in one aspect relates to a polynucleotide encoding a proexendin peptide. Such polynucleotides may be in the form of RNA or in the form of DNA, including cDNA, genomic DNA and synthetic DNA. In one embodiment of the invention, the proexendin transcript is encoded by nucleic acids 64-465 of the polynucleotide sequence of FIG. The nucleic acid encoding this particular proexendin peptide is a lizard-derived cDNA that encodes one transcript that encodes two peptides. The two peptides are the 40 amino acid peptide shown as amino acids 48-87 in FIG. 2 (note this), and the common dipeptidyl shown as amino acids 46 and 47 in FIG. A 45 amino acid peptide, shown as amino acids 1-45 in FIG. 2, linked to the N-terminus of exendin 4 peptide via a peptidase cleavage site. In addition, a common signal peptide sequence was found at the N-terminus of the transcript, indicating that the two encoded peptides were secreted from the cell. Thus, a 45 amino acid ENTP peptide would include a 23 amino acid signal peptide sequence and a 22 amino acid ENTP. By analogy with other polynucleotide sequences encoding peptide hormones such as glucagon and calcitonin, the ENTP peptide is expected to show biological activity as a peptide hormone. The invention is also based, in part, on the discovery that a mammalian homolog of proexendin exists. As shown in the Examples below, such homologs are expressed in at least mouse heart, skeletal muscle and pancreas. Given this information, as well as the demonstrated biological activity of exendin 4 on a variety of different mammalian cells, including humans, guinea pigs and rats, pro-exendin homologs can be found in any mammalian species There is. The proexendin nucleotide sequences of the present invention include: (B) a nucleotide sequence encoding the amino acid sequence shown in FIG. 2; (c) a high stringency condition [eg, 0.5M NaHPO _Four , 7% sodium dodecyl sulfate (SDS), hybridization with filter-bound DNA at 65 ° C. in 1 mM EDTA and washing at 68 ° C. in 0.1 × SSC / 0.1% SDS (Ausubel FM et al. (Eds.), 1989, Current Protocols in Molecular Biology, Vol. 1, Green Publishing Associates Inc. and John Wiley & Sons, Inc., New York, p. 2.10.3)] and hybridizes to the complement of the DNA sequence shown in FIG. Any nucleotide sequence encoding a highly equivalent gene product; and (d) under moderately stringent conditions, such as under moderately stringent conditions [eg, 42 ° C. in 0.2 × SSC / 0.1% SDS (Ausubel et al., 1989 supra)], an arbitrary nucleotide sequence that hybridizes with the complement of the DNA sequence encoding the amino acid sequence shown in FIG. 2 and encodes a functionally equivalent gene product. Functional equivalents of proexendin include natural proexendin present in other species and the functional activity of proexendin, whether natural or engineered (ie, Mutated proexendins that retain at least some of the peptides ENTP and processing to exendin, binding to exendin receptors, agonists of GLP-1, stimulating adenylate cyclase production, antagonists of GLP-1) . The invention also includes degenerate variants of sequences (a) to (d). The invention also includes nucleic acid molecules, preferably DNA molecules, which hybridize to the nucleotide sequences (a) to (d) described in the above paragraph and are therefore the complements thereof. The hybridization conditions may be highly stringent, as described above, or less stringent. When the nucleic acid molecule is a deoxyoligonucleotide (“oligo”), high stringency conditions include, for example, 37 ° C. (14 base oligo), 48 ° C. (17 base oligo), 55 ° C. in 6 × SSC / 0.05% sodium pyrophosphate. Washing at C (20 base oligo) and 60 C (23 base oligo). These nucleic acid molecules may encode, for example, a proexendin antisense molecule (and / or an antisense primer in an amplification reaction of the proexendin gene nucleic acid sequence) useful for proexendin gene regulation, or It may act as a sense molecule and / or primer. For regulation of the proexendin gene, such antisense sense techniques can be used, for example, to regulate suppression of cell function. In addition, sequences such as those described above can be used as part of a ribozyme and / or triple helix sequence, which is also useful for proexendin gene regulation. Furthermore, the nucleic acid molecule as described above can be used as a component of a diagnostic method for determining the tissue type of a tumor cell, for example. In addition to the proexendin nucleotide sequences described above, full-length proexendin cDNA or multiple gene sequences present in the same species without undue experimentation using molecular biology techniques well known in the art. Alternatively, homologues of the pro-exendin gene transcript present in other species can be identified and easily isolated. As shown in the examples below, pro-exendin transcripts are conserved between mammalian and reptile species. Indeed, isolating a polynucleotide encoding a novel proexendin homolog from a variety of mammalian cells, preferably human and mouse cells, known or suspected of containing proexendin. Can be. In particular, it can be isolated from cells of salivary gland, skeletal muscle, heart and pancreatic origin. In one embodiment, the invention provides proexendin polynucleotides or fragments thereof as a useful tool for identifying structurally related nucleic acids. For example, a nucleic acid library can be probed under low stringency hybridization conditions to identify genes at least about 40% homologous to the proexendin gene. Obviously, if you are looking for homologues from a particular species, such as humans, you have to find the right library. To facilitate isolation of the exendin-encoding homologue of the lizard exendin gene, the homologue desirably has 80% sequence identity to the lizard exendin gene at the nucleic acid level. More preferably, they are 90% identical and most preferably have at least 95% sequence identity when compared to the lizard proexendin gene. It will be apparent that increasing the stringency of the hybridization conditions will increase the sequence identity of the isolated homolog with the lizard exendin gene. For example, the exendin 3 gene can be isolated by using the nucleic acid sequence of the present invention to fish a cDNA library derived from the salivary gland of the lizard Heloderma horridum under high stringency conditions. To isolate an exendin-encoding homolog of the lizard proexendin gene, it is desirable, but not essential, to screen a library from pancreas, heart or skeletal muscle. Therefore, the present invention is directed not only to the gene encoding lizard xendin, but also to its structural homologues and in particular homologues encoding proteins having properties similar to exendin (eg, binding to the GLP-1 receptor). Including. Accordingly, the present invention provides a polynucleotide encoding proexendin, including the reptile proexendin gene, the lizard proexendin gene, and their mammalian homologs. Identification of novel proexendin homologues in related species may be useful in developing animal model systems that are more closely related to humans for drug discovery. For example, a cDNA library or genomic DNA library derived from an organism of interest can be screened by hybridization using the nucleotides described herein as a hybridization or amplification probe. In addition, similar techniques can identify genes at other loci in the genome that encode proteins with extensive homology to one or more novel domains of the proexendin gene product. It is. In the case of cDNA libraries, such a screening technique is capable of identifying clones derived from alternatively spliced transcripts in the same or different species. One of skill in the art can use the sequence of the exendin gene of at least about 15 nucleotides, preferably about 17 nucleic acids, to generate a probe useful for identifying a nucleic acid molecule encoding a proexendin homolog. Would be obvious. Of course, these sequences and the complete gene itself can also be used to clone proexendin-related genes by standard hybridization techniques. For example, DNA encoding another exendin (eg, another lizard exendin or a homolog of a mammal) can be obtained by applying a predetermined gene isolation or gene synthesis method. Screening is possible by performing filter hybridization using two types of filters. The labeled probe can include at least 15 to 30 base pairs of the proexendin nucleotide sequence shown in FIG. Hybridization wash conditions employed should be relatively low stringency if the cDNA library is derived from an organism different from the type of organism from which the labeled sequence was derived. For cloning of the mouse pro-exendin homolog using a reptile pro-exendin probe, hybridization can be performed overnight at 50 ° C., for example, in 1% SDS, 1 M NaCl, 2 μM EDTA, 1% BSA. The washing may be performed at 50 ° C. using 0.1 × SSC, 1% SDS. For cloning of the human homologue of the novel proexendin using the mouse proexendin probe, hybridization was performed using, for example, Church's buffer (7% SDS, 250 mM NaHPO4). _Four , 2 μM EDTA, 1% BSA) at 65 ° C overnight. Washing is performed at 65 ° C. using 2 × SSC, 0.1% SDS, and then at 65 ° C. using 0.1 × SSC, 0.1% SDS. Low stringency conditions are well known to those of skill in the art and will vary predictably depending on the particular organism from which the library and labeled sequences are derived. Guidance on such conditions include, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, NY; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, See NY. Isolation of exendin homologs typically involves extraction of total messenger RNA from a fresh source of pancreatic, cardiac or skeletal muscle tissue, followed by conversion of the message to cDNA and, for example, bacterial plasmids. Typically, this will entail creating a library in bacteriophage. These bacteriophages, which carry fragments of human DNA, are typically expanded by plating on a flora of susceptible E. coli bacteria to allow isolation of individual phage plaques or colonies. The DNA carried by the phage colonies is then immobilized on a hybridization membrane, typically of nitrocellulose or nylon type, and then radiolabeled (or otherwise) under carefully controlled conditions. The sequence is hybridized to identify the particular phage colony that carries the fragment of DNA of particular interest (in this case, a proexendin homolog). Subsequently, phages having the particular gene of interest are separated and purified from any other phage in the library to more easily characterize the foreign gene. Generally, the gene or a portion thereof is conveniently subcloned into a plasmid vector, particularly for complete sequencing of its DNA sequence. Thus, although the lizard proexendin gene has been provided herein, those skilled in the field of molecular biology will use mammalian pancreatic, heart or skeletal muscle cDNA or genomic libraries as probes to probe the lizard exendin gene or It will be appreciated that screening using the fragments can obtain mammalian homologues of the lizard exendin gene using standard molecular biology techniques. Further, by performing PCR using two degenerate oligonucleotide primers designed based on the amino acid sequence in the novel proexendin gene product disclosed herein, proexendin from the nucleic acid of the organism of interest can be obtained. Gene homologs can be isolated. Templates for this reaction are known or predicted to express alleles of such proexendin genes, eg, human or non-human cell lines or cell types (heart, skeletal muscle, pancreas). Cell type) can be cDNA obtained by reverse transcription of mRNA prepared from the same. The invention also encompasses mutant proexendins, peptide fragments of proexendins, truncated proexendin proteins, and nucleotide sequences encoding proexendin fusion proteins. Particularly preferred polynucleotides encode those functional domains of proexendin, such as a 47 amino acid ENTP peptide, a 45 amino acid ENTP peptide, a putative 23 amino acid secretion signal, a 22 amino acid ENTP peptide, and an exendin peptide 1- 40, 1-39 and 9-39. The nucleotides encoding the fusion protein can be an epitope tag to aid in the purification or detection of the resulting fusion protein, or a full-length proexendin fused to an unrelated protein or peptide, such as an enzyme, fluorescent protein, luminescent protein, etc. It may include, but is not limited to, truncated proexendin, or a peptide or fragment of proexendin. The proexendin gene sequence can also be used to isolate mutant proexendin gene alleles. Such mutated alleles can be isolated from individuals known to have a genotype associated with the symptoms of metabolic disorders, or from individuals proposed to have this type of genotype. In addition, such proexendin gene sequences can be used to detect proexendin gene regulation (eg, promoter or promoter / enhancer) defects that may be involved in metabolic disorders. In addition, nucleotides encoding putative secretory signal sequences can be fused in reading frame to nucleotides encoding a heterologous gene product, and thus be used to direct secretion of such gene products. Can be. The present invention also provides (a) a DNA vector comprising any of the proexendin coding sequences and / or their complements (ie, antisense); and (b) a regulatory vector that directs the expression of the proexendin coding sequence. A DNA expression vector comprising any of the coding sequences operably linked to an element; and (c) the DNA sequence operably linked to a regulatory element that directs the expression of the proexendin coding sequence in a host cell. Genetically engineered host cells containing any of the coding sequences. With the nucleic acid encoding proexendin in hand, the proexendin gene product can be used for in vitro transcription and integration of the DNA into an appropriate expression vector and a suitable host (e.g., bacteria such as E. coll, yeast or Can be produced in several ways, including expression in mammalian cells. A substance is said to be "isolated" or "substantially free of natural contaminants" if it is substantially purified from substances that normally coexist in nature. It should be noted. The term "recombinant polynucleotide" as used herein means that the polynucleotide is not in its natural environment, e.g., that the polynucleotide is operably linked to a heterologous nucleic acid sequence. It should be noted that Various gene expression systems have been adapted to be used with these hosts, and they are now commercially available. Any one of these systems can be selected to express DNA encoding exendin. The expression vector is selected to provide a transformed cell line that transiently or stably expresses the DNA encoding proexendin. In the case of transient expression, the host cell is typically transformed with an expression vector having an origin of replication functional in a mammalian cell. In the case of stable expression, such an origin of replication is not required, but the vector does not require a gene encoding a product that confers a survival advantage to the transformed cells, such as neomycin resistance, to allow for their selection. Will carry the encoding gene (in this case, the transformed cells will be plated on media supplemented with neomycin). These systems are typically available in the form of a plasmid vector, and incorporate an expression cassette whose functional components include expression regulatory sequence constituent DNA. This expression cassette is host-recognizable and is ligated 5 'to the pro-exendin-encoding DNA to enable expression of the pro-exendin-encoding DNA. This system further incorporates a DNA sequence linked 3 'to the proexendin-coding region to terminate expression. Thus, when expressed in a selected mammalian cell host, a recombinant DNA expression construct is obtained in which the proexendin-encoding DNA is linked to an expression regulatory DNA sequence recognized by the host, and this recombinant DNA expression construct is obtained. The construct includes a 5 'region of the proexendin-encoding DNA to drive expression and a 3' region to terminate expression. Various recombinant DNA expression systems that can be used to achieve expression of exendin-encoding DNA in mammalian cells include promoters for viruses that infect mammalian cells [eg, cytomegalovirus (CMV), Rous sarcoma virus (RSV), simian virus (SV40), mouse mammary adenocarcinoma virus (MMTV), and other promoters]. Retroviral LTR-like promoters, insect cell promoters (eg, controlled by temperature), and promoters isolated from Drosophila, and mammalian gene promoters (eg, controlled by heavy metals; , Metallothionein gene promoters and other steroid-inducible promoters) are also useful in driving expression. The novel proexendin polypeptides and peptide fragments, mutant, truncated or truncated proexendins, and proexendin fusion proteins can be prepared for a variety of uses, including For example, production of antibodies as reagents in diagnostic assays; cell metabolism as reagents in assays to screen for compounds that can be used to treat metabolic disorders, and as pharmaceutical reagents useful in treating metabolic disorders such as diabetes Identification of other cellular gene products involved in the regulation of The proexendin amino acid sequence of the present invention comprises the amino acid sequence shown in FIG. 2 (SEQ ID NO: 2). In addition, other species of proexendin are encompassed by the present invention. In fact, any protein encoded by the above-described proexendin nucleotide sequence is within the scope of the invention, as long as it includes either the ENTP sequence or the novel carboxyl terminus of exendin. In particular, when used to detect the presence and / or location of exendin and ENTP (eg, in toxicants, pancreas, heart or skeletal muscle cells), the present invention provides, in another embodiment, an ENTP peptide or exendin Antibodies. Antibodies to the proexendin peptide sequence (including antibodies specific for the domain of proexendin) are useful for a variety of purposes. For example, proexendin (including the ENTP peptide) can be used as a marker for tissue-specific activity. Endocrine tumors that secrete abnormal amounts of proexendin or fragments of proexendin can be detected by assaying for circulating levels of proexendin or proexendin fragments. Specifically, proexendin is known to be expressed by pancreatic, heart and skeletal muscle tissues. In addition, organ or tissue failure of the proexendin-expressing tissue can be manifested by a change or decrease in the amount of detectable proexendin. In addition, the above-described antibodies are useful for detecting the presence, absence, and level of toxic peptides in humans that have been bitten by reptiles and have entered the body. Further, the antibodies described above are useful for neutralizing toxic proteins. To raise the above antibodies, the ENTP gene product, exendin, or immunogenic fragments thereof, produced as described above in a microorganism or mammalian host cell, or by standard peptide synthesis techniques, can be used. Specifically, a 45 amino acid ENTP or a 22 amino acid ENTP, or an immunogenic fragment thereof can be used for this. The standard immunization protocol is as follows. Typically, the peptide is formulated in PBS, but an adjuvant (eg, Freund or alum) may be mixed with the immunogen. Usually, for peptides with a molecular weight of less than 10 kd, the peptide is bound to a carrier such as keyphor limpet hemocyanin. In one useful immunization protocol, test animals are typically injected subcutaneously with an immunogen containing the peptide in an order of 0.5-1 mg per bolus, and after about 2-3 weeks the peptide (compounded in PBS) One) intramuscular booster injection with 100-200 μg. This intramuscular injection may be repeated once after a few days if found necessary from the test bleeds. One source of antibodies to the proexendin gene product of interest or a fragment thereof is from the blood of an animal immunized with the immunogen described above. Alternatively, when producing monoclonal antibodies, immunocompetent cells (eg, splenocytes) are removed from the immunized animal and fused with myeloma cells using hybridoma technology. Monoclonal antibodies are particularly preferred for generating antibodies specific for a single epitope. Next, the fusion product is screened by culturing in a selective medium, the antibody-producing cells are collected, and the cells are subsequently grown to collect the antibody. The recovered antibody can then be covalently linked to a detectable label (eg, a radioactive label, an enzyme label, a luminescent label, etc.). This is done using the linker technology established for this purpose. Animal model systems that elucidate the physiological and behavioral roles of proexendin gene products will either increase or decrease the activity of the encoded peptide or alter the amino acid sequence of the expressed proexendin peptide of interest By creating various transgenic animals by various techniques. Examples of these techniques include (1) encoding a proexendin peptide by microinjection, electroporation, transfection with a retrovirus, or other means well known to those of skill in the art to produce transgenic animals. Insertion of a normal or mutated form of DNA into a suitable fertilized embryo, or (2) those having a native locus in the transgenic animal to modify the expression regulation or structure of these peptide sequences. , Or homologous recombination of a human or animal version of the normal or mutant form of the Techniques for homologous recombination are well known in the art. This technique replaces the native gene with the inserted gene, and therefore cannot express the native proexendin peptide, but can, for example, produce an animal in which the inserted mutant proexendin peptide is expressed. Useful. In this case, the native proexendin peptide in the animal genome has been replaced by recombination, so that the peptide is underexpressed. Microinjection adds (adds) a gene to the genome, rather than removing it from the genome, and is therefore useful for producing animals that express their own pro-exendin peptide and the added pro-exendin peptide As a result, the desired proexendin or peptide is overexpressed. One means that can be used to generate transgenic animals, such as mice, is as follows. Female mice are mated and the resulting fertilized eggs are removed from the oviduct. The fertilized eggs are stored in a suitable medium such as M2 medium. The vector containing the DNA or cDNA encoding the proexendin peptide is subjected to cesium chloride purification by methods well known in the art. An inducible promoter can be fused to the coding region of DNA to provide an experimental means of regulating transgene expression. Alternatively, or additionally, a tissue-specific regulatory element can be fused to the coding region to allow for tissue-specific expression of the transgene. DNA (suitably in a buffer solution) is introduced into a microinjection needle (which can be made from capillary material using a pipette puller), and a fertilized egg to be injected is placed on a glass slide having a recess. . Insert the needle into the pronucleus of the fertilized egg and inject the DNA solution. The infused fertilized eggs are then transferred to the oviduct of a pseudopregnant mouse (a mouse maintained in pregnancy by stimulation with the appropriate hormone, but not actually pregnant). The fertilized egg moves to the uterus, implants, develops and gives birth. As noted above, microinjection is not the only method for inserting DNA into egg cells, and is used herein for illustrative purposes only. Example 1 Isolation of proexendin gene Total cellular RNA from salivary glands of the lizard Heloderma suspect um was isolated by the acid guanidium thiocyanate method (Chom czynski, P. et al., 1987, Anal. Chem. 169, 158-159). For first strand cDNA synthesis, 5 μg of whole cell RNA was reverse transcribed using a SuperScript Preamplification system (Gibco-Bethesda Research Laboratories (BRL), Gaithersburg, MD). Subsequently, an aliquot (0.1 volume) of the first strand cDNA was amplified by PCR (35 cycles, annealing temperature 50 ° C, extension temperature 72 ° C). To isolate lizard exendin, degenerate primers were used for LE (A); 5'-GCG GAA TTC AYG GNG ARG GNA CNTT-3 'and LE (B); 5'-CGC GGA TCC RTT YTT NAR CCA YTC- 3 '. The amplification product was cloned into a TA cloning vector pCRII (Invitrogen, San Diego, CA) and sequenced. A PCR product of the expected size (95 bp) was obtained from RNA derived from the salivary gland of the lizard, and sequencing of this partial cDNA revealed that it encodes the sequence of exendin-4. To isolate full-length exendin cDNA, a lizard salivary gland cDNA library was generated and then screened with a partial exendin cDNA probe. H.suspectum cDNA libraries were generated from poly (A) and mRNA isolated from adult lizard salivary glands. This cDNA library was constructed at 1 × 10 5 at the EcoRI site of a pcDNAII vector (Invitrogen, SanDiego, Calif.). ⁶ Consisting of two independent recombinant phagemids. This cDNA library was screened with the 95 bp nucleotide sequence EcoRI / BamHI [α32P] -dATP-labeled lizard cDNA generated by RT-PCR just as described above. From this library, a single class of exendin cDNA clones encoding exendin (amino acids 48-87 in FIG. 1) and the 45 amino acid peptide N-terminal to the exendin sequence (amino acids 1-45 in FIG. 1) were isolated. . This 45 amino acid peptide is named exendin N-terminal peptide (ENTP). The ENTP sequence is followed by a dibasic pair of amino acids characterized by a prohormone convertase cleavage site, ie, lysine and arginine, followed by a 40 amino acid exendin sequence. Example 2 Identification of a Mammalian Homolog of Exendin Lizard exendin nucleotide probes can be used to obtain mammalian exendin cDNA by standard molecular biology techniques. In this example, the lizard exendin probe was ³² P-labeled and used for Northern blot analysis (hybridization was performed at 50 ° C. in 1 M NaCl, 1% SDS, washing was performed at 50 ° C. in 0.1 × SSC, 1% SDS) and the corresponding mouse exendin mRNA transcription. Products were detected in various mouse tissues. Mouse exendin mRNA transcript (about 1.2 kb in size) was detected in RNA from mouse heart. Mouse exendin mRNA (about 1.0 kb in size) is also detected in RNA from skeletal muscle, and a mouse exendin RNA transcript of about 1.0 kb can be detected in mouse pancreatic RNA. One of skill in the art would be able to screen not only mouse exendin cDNA, but also other mammalian exendin cDNA, including full-length nucleotide mammalian exendin cDNA, from these tissues using a lizard exendin probe to screen a cDNA library. Or by utilizing RT-PCR. The present invention is not to be limited in scope by the specific embodiments described herein. This embodiment is to be construed as merely exemplary of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications fall within the scope of the appended claims.

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Claims (1)

[Claims] 1. A polynucleotide encoding proexendin, which is recombinant   Or said polynucleotide in an isolated form. 2. 2. The polynucleotide of claim 1 wherein said polynucleotide has the sequence of FIG.   Chid. 3. A recombinant polynucleotide encoding the peptide of FIG. 4. Encodes amino acids 1-47, 1-23, 24-47, 24-45, 48-87 or 48-86 of FIG.   Recombinant polynucleotide. 5. A polynucleotide according to any one of claims 1 to 4 under stringent conditions.   Recombinant polynucleotides that hybridize to nucleotides or their complements   De. 6. 6. The recombinant polypeptide of claim 5, wherein said recombinant polypeptide is derived from a lizard, Heloderma horridum.   A recombinant polynucleotide according to any one of the preceding claims. 7. 6. A method as claimed in any one of claims 1 to 5 under moderate stringency conditions.   And a mammalian DNA that hybridizes with the polynucleotide or the complement thereof.   A recombinant polynucleotide obtained from 8. The recombinant polypeptide encoding the peptide having the amino acid sequence of amino acids 1-47 in FIG.   Renucleotide. 9. The recombinant polypeptide encoding the peptide having the amino acid sequence of amino acids 1-45 in FIG.   Renucleotide. Ten. Recombination encoding a peptide having the amino acid sequence of amino acids 24-45 of FIG.   Polynucleotide. 11． Recombination encoding a peptide having the amino acid sequence of amino acids 48-87 of FIG.   Polynucleotide. 12． Recombination encoding a peptide having the amino acid sequence of amino acids 48-86 of FIG.   Polynucleotide. 13. A substantially purified peptide having the amino acid sequence shown in FIG. 14. A substantially purified peptide having the amino acid sequence of amino acids 1-45 of FIG. 15． A substantially purified peptide having the amino acid sequence of amino acids 24-45 of FIG. . 16． A substantially purified peptide having the amino acid sequence of amino acids 48-87 of FIG. . 17． A method comprising administering a peptide according to any one of claims 13 to 16.   A method of inducing an immune response in an object. 18． 19. The method of claim 18, wherein said peptide is amino acid residues 24-45 of FIG. 19. An immune response is induced in an animal by the method of claim 17 and the animal is   Isolating an antibody that specifically recognizes a xendin peptide.   A method for producing an antibody specific to a sendin peptide.