CN1286698A - Human angiotonin II/vasopressin receptor like gene - Google Patents

Abstract

CBDAKD01 polpeptides and polynucleiotides and methods for producing such polypeptides by recombinant techniques are disclosed. Also disclosed are methods for utilizing CBDAKD01 polypeptides and polynucleotides in the design of protocols for the treatment of hypertension, heart disease, cancer, and kidney disease, among others, and diagnostic assays for such conditions.

Description

human angiotensin Ⅱ/vasopressin receptor-like gene

field of invention

The present invention relates to newly identified polynucleotides, polypeptides encoded thereby and uses of these polynucleotides and polypeptides and their production. More specifically, the polynucleotides and polypeptides of the present invention are related to the angiotensin and/or vasopressin receptor family (hereinafter referred to as CBDAKD01). The present invention also relates to inhibiting or activating the action of these polynucleotides and polypeptides.

Background of the invention

The rat angiotensin/vasopressin receptor (AII/AVP) is a dual receptor that can bind both angiotensin II and vasopressin. This suggests a well-documented precedent for the angiotensin and/or vasopressin receptor family as therapeutic targets. Clearly, there is a need to identify and characterize other members of the angiotensin and/or vasopressin receptor family that play a role in preventing, alleviating and correcting dysfunction or disease including, but not limited to, hypertension, Heart disease, cancer and kidney disease.

Summary of the invention

One aspect of the invention relates to CBDAKD01 polypeptides and recombinant materials and methods for their production. In another aspect, the present invention relates to methods of using CBDAKD01 polypeptides and polynucleotides, including treating diseases such as hypertension, heart disease, cancer and kidney disease. In yet another aspect, the invention relates to methods of using the materials provided herein to identify agonists and antagonists and using the identified compounds to treat symptoms associated with CBDAKD01 imbalance. Finally, the present invention relates to diagnostic assays for examining diseases associated with abnormal activity or levels of CBDAKD01. DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

To facilitate the understanding of terms commonly used in this specification, the following definitions are provided.

"CBDAKD01" generally refers to a polypeptide having the amino acid sequence shown in Sequence 2 or an allelic variant thereof.

"CBDAKD01 activity or CBDAKD polypeptide activity" or "CBDAKD01 or CBDAKD01 polypeptide biological activity" refers to the metabolic or physiological function of CBDAKD01, including similar activity or enhanced activity or activity with less undesired side effects. Said activity also includes the antigenic and immunogenic activity of CBDAKD01.

"CBDAKD01 gene" refers to the polynucleotide having the nucleotide sequence shown in Sequence 1 or its allelic variant and/or its complement.

"Antibody" herein includes monoclonal and polyclonal antibodies, chimeric, single chain and humanized antibodies and Fab fragments, including the products of Fab or other immunoglobulin expression libraries.

"Isolated" means altered "by the hand of man" from its natural state. An "isolated" composition or substance has been altered or removed from its original environment or has been removed and altered if it occurs in nature. For example, a polynucleotide or polypeptide present in a living body is not "isolated" when the term is used herein, but the same polynucleotide or polypeptide that has been separated from coexisting substances in a natural state is "isolated".

"Polynucleotide" generally refers to polyribonucleotides or polydeoxyribonucleotides, which may be unmodified or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single- and double-stranded DNA, DNA mixed with single- and double-stranded regions, single- and double-stranded RNA, mixed single- and double-stranded regions of RNA, hybrids containing DNA and RNA A molecule that can be single-stranded or more commonly double-stranded or a mixture of single- and double-stranded regions. Furthermore, "polynucleotide" also refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The term "polynucleotide" also includes DNA or RNA containing one or more modified bases, as well as DNA or RNA whose backbone has been modified for stability or for other reasons. "Modified" bases include unusual bases such as tritylated bases and inosine. A variety of modifications can be made to DNA and RNA, thus, "polynucleotide" includes chemically, enzymatically or metabolically modified forms of polynucleotides commonly found in nature, as well as chemical forms of DNA and RNA characteristic of viruses and cells. "Polynucleotide" also includes relatively short polynucleotides, commonly referred to as oligonucleotides.

"Polypeptide" refers to any peptide or protein comprising two or more amino acids interconnected by peptide bonds or modified peptide bonds, ie, peptide isosteres. "Polypeptide" includes both short chains (often called peptides) and long chains (often called proteins) of oligopeptides or oligomers. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. A "polypeptide" includes an amino acid sequence modified by natural methods such as post-translational processing or by chemical modification techniques known in the art. These modifications are well described in basic textbooks and monographs, as well as in numerous research papers. Modifications can occur anywhere in the polypeptide, including the peptide backbone, amino acid side chains, and amino or carboxyl termini. Modifications of the same type may be present to the same or different degrees at different positions in a given polypeptide. And a given polypeptide may contain many types of modifications. Polypeptides may be branched due to ubiquitination, or may be circular with or without branching. Cyclic, branched and branched cyclic polypeptides may arise as a result of post-translational natural processes or synthetic methods. Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent conjugation of flavins, covalent conjugation of heme, covalent conjugation of nucleotides or nucleotide derivatives, lipids or Covalent conjugation of lipid derivatives, covalent conjugation of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, cystine formation, pyroglutamine Acid formation, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, isoglutarylation , racemization, selenoylation, sulfation, transfer RNA-mediated incorporation of amino acids into proteins such as arginylation and ubiquitination (see e.g. Proteins: Structure and Molecular Properties 2nd ed. , T.E.Creighton, W.H.Freeman and Company, New York, 1993 and Wold, F., "Posttranslational Protein Modifications: Prospects and Perspectives", pp. 1-12, "Posttranslational Covalent Modifications of Proteins", B.C.Johnson, Ed., Academic Press, NewYork, 1983; Seifter et al., "Analysis of protein modifications and nonprotein cofactors", MethEnzymol (1990) 82:626-646 and Rattan et al., "Protein synthesis: post-translational modifications and aging", Ann NY Acad Sci (1992) 663:48-62).

"Variant" refers to a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, but retains essential properties. Typically, a polynucleotide variant differs in nucleotide sequence from a reference polynucleotide. Changes in the nucleotide sequence of a variant may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. As described below, nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. Typically, a polypeptide variant differs in amino acid sequence from a reference polypeptide. Typically, the differences are limited such that the sequences of the reference polypeptide and the variant are generally very similar and identical in many regions. A variant and a reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A substituted or inserted amino acid residue may or may not be an amino acid encoded by the genetic code. Variants of a polynucleotide or polypeptide may be naturally occurring, such as allelic variants, or non-naturally occurring. Non-naturally occurring polynucleotide and polypeptide variants can be prepared by mutagenesis techniques or direct synthesis.

"Identity" is known in the art as the relationship between two or more polypeptide sequences or polynucleotide sequences determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between polypeptide or polynucleotide sequences as determined by the match between strands of these sequences. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology, Lesk, A.M., ed. Oxford University Press, New York, 1988; Computational Biology: An Introduction and Genome Engineering, Smith D.W., ed., Academic Press, New York, 1993; Computational Analysis of Sequence Data, Part 1, Griffin A.M., and Griffin H.G., eds., Humana Press, NewJersey , 1994; "Sequence Analysis in Molecular Biology", von Heiinje, G., Academic Press, 1987; "Primers for Sequence Analysis", Gribskov, M. and Devereux, J., eds., M StocktonPress, NeW York, 1991 ; Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). Preferably, methods for determining identity are designed to maximize the match between the sequences tested. Furthermore, methods to determine identity and similarity have been codified in publicly available computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, but are not limited to, the GCG package (Devereux, J., et al., Nucleic Acids Research (1984) 12(1):387), BLASTP, BLASTN, FASTA (Atschul, S.F. et al., J Molec Biol (1990) 215:403). The BLAST X program is available from NCBI or other sources (BLAST Handbook, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990).The well-known Smith Waterman algorithm can also be used to determine identity.

The preferred parameters of polypeptide sequence comparison include: 1) algorithm: Needleman and Wunsch, J.Mol Biol.48:443-453 (1970) comparison matrix (Comparison matrix): BLOSSUM62, from Hentikoff andHentikoff, Proc.Natl.Acad.Sci. USA.89: 10915-10919 (1992) Gap Compensation: 12 Gap Length Compensation: 4 One program that can apply these parameters is the gap program provided by Genetics Computers Group, Madison WI. The above parameters are the default parameters for peptide comparisons (end gaps are not compensated).

The preferred parameters of polynucleotide comparison include: 1) algorithm: Needleman and Wunsch, J.Mol Biol.48: 443-453 (1970) comparison matrix (Comparison matrix): matching=+10, mismatch=0 gap compensation: 50 Gap Length Compensation: 3 One program that can use these parameters is the gap program provided by the Genetics Computers Group, Madison WI. The above parameters are the default parameters for nucleic acid comparisons.

Preferred polynucleotide embodiments also include isolated polynucleotides comprising at least 50, 60, 70, 80, 85, 90, 95, 97, or 100% identity to a polynucleotide reference sequence of SEQ ID NO: 1, wherein said The sequence may be identical to the reference sequence of SEQ ID NO: 1, or comprise a number of nucleotide changes compared to the reference sequence, wherein the changes are selected from at least one nucleotide deletion, substitution (including transitions and transversions) or insertion, wherein The alterations may occur at the 5' or 3' end of the reference polynucleotide sequence or anywhere in between, interspersed respectively between nucleotides of the reference sequence, or in one or more contigs distributed in the reference sequence. The number of nucleotide changes is determined by multiplying the total number of nucleotides in sequence 1 by the corresponding percent identity and subtracting the result from the total number of nucleotides in sequence 1, or by:

n _n ≤ X _n -(X _n y)

where n _n is the number of nucleotide changes, x _n is the total nucleotide number of sequence 1, and the y value is 0.50 at 50%, 0.60 at 60%, 0.70 at 70%, and 80% at 0.80, 0.85 at 85%, 0.90 at 90%, 0.95 at 95%, 0.97 at 97%, 1.00 at 100%, where if the results of x _n and y are not integers, then the It rounds to the nearest whole number and subtracts from x _n . Changes in the polynucleotide sequence encoding the polypeptide of Sequence 2 will produce nonsense, missense or frameshift mutations in its coding sequence, so that the polypeptide encoded by the altered polynucleotide will change.

Preferred polypeptide embodiments also include isolated polypeptides comprising at least 50, 60, 70, 80, 85, 90, 95, 97, or 100% identity to a polypeptide reference sequence of Sequence 2, wherein said sequence may be identical to that of Sequence 2. The reference sequence is identical, or comprises a certain number of amino acid changes compared to the reference sequence, wherein the change may be selected from at least one amino acid deletion, substitution (including conservative and non-conservative substitutions) or insertion, wherein the change may occur in the reference polypeptide The amino or carboxyl termini of the sequence, or anywhere in between, respectively, are interspersed among the amino acids of the reference sequence, or are interspersed in one or more contigs in the reference sequence. The number of amino acid changes is determined by multiplying the total number of amino acids in Sequence 2 by the corresponding percent identity and subtracting the result from the total number of amino acids in Sequence 2, or by:

n _a ≤ x _a -(x _a y)

Among them, n _a is the number of amino acid changes, x _a is the total amino acid number of sequence 2, and the y value is 0.50 at 50%, 0.60 at 60%, 0.70 at 70%, 0.80 at 80%, and 0.80 at 85%. 0.85 at 90%, 0.90 at 90%, 0.95 at 95%, 0.97 at 97%, 1.00 at 100%, where the results of x _a and y are rounded to the nearest if they are not integers Integer of , and then subtracted from x _a . Polypeptides of the invention

In one aspect, the invention relates to CBDAKD01 polypeptides (or CBDAKD01 proteins). The CBDAKD01 polypeptide includes the polypeptide of Sequence 2, and the polypeptide comprising the amino acid sequence of Sequence 2, and the polypeptide comprising an amino acid sequence which is at least 80% identical to the entire length of Sequence 2, preferably at least 90% identical to Sequence 2, More preferably at least 95% identity. Sequences that are at least 97-99% identical are highly preferred. CBDAKD01 polypeptides also include polypeptides whose amino acid sequence is at least 80% identical to the full-length polypeptide having the amino acid sequence of Sequence 2, preferably at least 90% identical to Sequence 2, more preferably at least 95% identical. Sequences that are at least 97-99% identical are highly preferred. Preferred CBDAKD01 polypeptides have at least one CBDAKD01 biological activity.

The CBDAKD01 polypeptide can be in the form of a "mature" protein, or it can be part of a larger protein, such as a fusion protein. It is often advantageous to include additional amino acid sequences, including secretory or leader sequences, prosequences, sequences to aid in purification such as polyhistidine residues, or additional sequences to aid in stability during recombinant production.

The present invention also includes CBDAKD01 polypeptide fragments. A fragment is one whose amino acid sequence is completely identical to a part of the amino acid sequence of the CBDAKD01 polypeptide, but does not have the entire amino acid sequence of CBDAKD01. As with the CBDAKD01 polypeptide, fragments may be independent or included in a larger polypeptide as part or a region, preferably as a single contiguous region. Representative examples of polypeptide fragments of the invention include, for example, fragments from approximately amino acids 1-20, 21-40, 41-60, 61-80, 81-100, and 101 to the end of the CBDAKD01 polypeptide. "About" includes several, 5, 4, 3, 2 or 1 amino acid more or less than the specified range at either or both ends.

Preferred fragments include, for example, truncated polypeptides having the amino acid sequence of the CBDAKD01 polypeptide, but missing a stretch of contiguous residues including the amino-terminus or a stretch of contiguous residues including the carboxy-terminus, or missing two contiguous residues, one stretch including the amino terminus, one segment including the carboxyl terminus. Fragments characterized by structural or functional properties are also preferred, for example fragments comprising the following structures: α-helices and α-helix forming regions, β-sheets and β-sheet forming regions, turns and turn forming regions, coils and coils Forming region, hydrophilic region, hydrophobic region, alpha amphipathic region, beta amphiphilic region, flexible region, surface forming region, substrate binding region and high antigenic index region. Other preferred fragments are biologically active fragments. Biologically active fragments are fragments that mediate the activity of CBDAKD01, including fragments with similar or higher activities, or fragments with lower undesired activities. Also included are fragments that are antigenic or immunogenic in animals, especially humans.

Preferably, all of these polypeptide fragments retain the biological activity of CBDAKD01, including antigenic activity. Variants of the given sequences and fragments are also part of the invention. Preferred variants are those with conservative amino acid substitutions in the reference, ie, residues in the reference are replaced by other amino acids with similar properties. Typical such substitutions include substitutions between Ala, Val, Leu, and Ile; substitutions between Ser and Thr; substitutions between acidic residues Asp and Glu; substitutions between Ash and Gln; basic residues Lys and Arg; or between the aromatic residues Phe and Tyr. Particularly preferred are variants in which several, 5-10, 1-5 or 1-2 amino acids are substituted, deleted or added, or combinations of these.

The CBDAKD01 polypeptides of the present invention can be prepared by any suitable means. These polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Methods for preparing these polypeptides are well known in the art. polynucleotides of the invention

Another aspect of the invention relates to CBDAKD01 polynucleotides. CBDAKD01 polynucleotides include isolated polynucleotides encoding CBDAKD01 polypeptides and fragments as well as polynucleotides closely related thereto. More specifically, the CBDAKD01 polynucleotide of the present invention includes a polynucleotide comprising the nucleotide sequence of Sequence 1, a CBDAKD01 polypeptide encoding Sequence 2, and a polynucleotide having a specific sequence of Sequence 1. CBDAKD01 polynucleotides also include polynucleotides that contain a nucleotide sequence that is at least 82% identical to the full-length nucleotide sequence of the CBDAKD01 polypeptide encoding Sequence 2, and polynucleotides that contain a nucleotide sequence that is at least 82% identical to the entire length of Sequence 1. 82% of the nucleotide sequences are polynucleotides. Polynucleotides having at least 90% identity are preferred, and polynucleotides having at least 95% identity are especially preferred. Polynucleotides that are at least 97% identical are highly preferred, polynucleotides that are at least 98-99% identical are more preferred, and polynucleotides that are at least 99% identical are most preferred. CBDAKD01 polynucleotides also include nucleotide sequences whose identity is sufficient to hybridize to the nucleotide sequence in Sequence 1 under conditions for amplification or as a probe or label. The present invention also provides a polynucleotide complementary to the CBDAKD01 polynucleotide.

The CBDAKD01 of the present invention is structurally related to other proteins of the angiotensin and/or vasopressin receptor family, which is confirmed by the results of sequencing the cDNA encoding human CBDAKD01 in Table 1 (SEQ ID NO: 1). The cDNA sequence of Sequence 1 contains an open reading frame (nucleotides 47-1588) encoding the 514 amino acid polypeptide of Sequence 2. The amino acid sequence of table 2 (sequence 2) 485 amino acid residues and rat angiotensin/vasopressin receptor (AII/AVP) (N.Ruiz-Opazo, et al., Nature, Med.1: 074- 1081, 1995) about 30% identity (FASTA). The nucleotide sequence of table 1 (sequence 1) 489 nucleotide residues and rat angiotensin/vasopressin receptor (AII/AVP) (N.Ruiz-Opazo, et al., Nature, Med. 1:074-1081, 1995) about 57.9% identity (FASTA). Therefore, CBDAKD01 polypeptides and polynucleotides of the present invention are expected to have similar biological functions/properties to their homologous polypeptides and polynucleotides, and their uses will be apparent to those skilled in the art.

^a人CBDAKD01核苷酸序列(序列1)。Table ^1a

^a Nucleotide sequence of human CBDAKD01 (SEQ ID NO: 1).

^b人CBDAKD01氨基酸序列(序列2)。Table ^2b

^b Human CBDAKD01 amino acid sequence (SEQ ID NO: 2).

The polynucleotide encoding CBDAKD01 of the present invention can be obtained by standard cloning and screening from a cDNA library derived from mRNA derived from human cord blood cells using expressed sequence tag (EST) analysis (Adams, M.K., et al., Science (1991 ) 252: 1651-1656; Adams, M.D., et al., Nature, (1992) 355: 632-634; Adams, M.D., et al., Nature (1995) 377 Supp: 3-174). The polynucleotides of the invention may also be derived from natural sources such as genomic DNA libraries, or may be synthesized using well known commercially available techniques.

The nucleotide sequence of the CBDAKD01 polypeptide encoding sequence 2 may be identical to the polypeptide encoding sequence (nucleotide 47-1588 of sequence 1) in Table 1, or may be formed due to the redundancy (degeneracy) of the genetic code but still encode sequence 2 sequence of the polypeptide.

When the polynucleotide of the present invention is used for recombinant production of CBDAKD01 polypeptide, the polynucleotide may include the coding sequence itself of the mature polypeptide or its fragment; the sequence of the mature polypeptide or fragment coding sequence fused with other coding sequences in frame, other coding sequences For example the coding sequence of a leader or secretory sequence, a pre or pro or prepro protein sequence or other fusion polypeptide portion. For example, a tag sequence may be encoded to facilitate purification of the fusion polypeptide. In some preferred embodiments of this aspect of the invention, the tag sequence is a 6-histidine peptide or an HA tag, the former provided in the pQE vector (Qiagen, Inc.), as discussed in Gentz et al., Proc Natl Acad Sci USA (1989) 86: 821-824. A polynucleotide may also contain noncoding 5' or 3' sequences, such as transcribed, untranslated sequences, splicing and polyadenylation signals, ribosome binding sites, and sequences that stabilize mRNA.

A further preferred embodiment is a polynucleotide encoding a variant of CBDAKD01, said variant comprising the amino acid sequence of the CBDAKD01 polypeptide in Table 2 (Sequence 2), wherein several, 5-10, 1-5, 1 - 3, 1-2 or 1 amino acid residues or a combination of these.

The present invention also relates to polynucleotides that hybridize to the above sequences. In particular, the present invention relates to polynucleotides that hybridize to the aforementioned polynucleotides under stringent conditions. Herein, "stringent conditions" means that hybridization occurs only when the identity between the sequences is at least 80%, preferably at least 90%, more preferably at least 95%, most preferably 97-99%.

The polynucleotides of the present invention are identical or sufficiently identical to the nucleotide sequences of Sequence 1 or fragments thereof, and thus can be used as hybridization probes for cDNA and genomic DNA to isolate full-length cDNA and genomic clones encoding CBDAKD01, and can also isolate cDNA and genomic cloning of other genes with high sequence similarity to the CBDAKD01 gene, including genes encoding homologues and orthologs in species other than humans. These hybridization techniques are known to those skilled in the art. Generally, these nucleotide sequences have 80% identity with the reference sequence, preferably 90% identity, more preferably 95% identity. Probes generally comprise at least 15 nucleotides. Preferably these probes comprise at least 30 nucleotides or at least 50 nucleotides. Particularly preferred probes are 30 to 50 nucleotides.

In one embodiment, the method for obtaining polynucleotides encoding CBDAKD01 polypeptides including homologues or orthologs of species other than humans comprises the following steps: using a labeled probe having sequence 1 or a fragment thereof in stringent hybridization Screening of suitable libraries under conditions; isolation of full-length cDNA and genomic clones containing the polynucleotide sequence. Therefore, on the other hand, the CBDAKD01 polynucleotide of the present invention also includes a nucleotide sequence that hybridizes to the nucleotide sequence of Sequence 1 or a fragment thereof under stringent conditions. The CBDAKD01 polypeptide also includes the following polypeptides, the amino acid sequence of which is encoded by the nucleotide sequence obtained under the above hybridization conditions. These hybridization techniques are well known to those skilled in the art. The stringent hybridization conditions are as described above, or in other words the following conditions: incubate overnight at 42°C in the following solution, and then wash the filter membrane with 0.1×SSC at about 65°C, said solution includes 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate and 20 μg/ml denatured sheared salmon sperm DNA.

The polynucleotides and polypeptides of the present invention can be used as research reagents and materials to find methods for treating and diagnosing animal and human diseases. vector, host cell, expression

The invention also relates to vectors containing one or more polynucleotides of the invention, host cells genetically engineered to contain the vectors of the invention, and the production of polypeptides of the invention by recombinant techniques. Using RNA derived from the DNA constructs of the invention, cell-free translation systems can be used to produce these proteins.

For recombinant production, host cells can be genetically engineered to contain an expression system of a polynucleotide of the invention, or a portion thereof. Introduction of polynucleotides into host cells can be accomplished by a variety of methods described in standard laboratory manuals, such as Davis et al., Fundamental Methods of Molecular Biology (1986) and Sambrook et al., Molecular Cloning : Laboratory Manual "Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), the method includes calcium phosphate transfection, DEAE-dextran mediated transfection, transposition, microinjection, Cationic lipid-mediated transfection, electroporation, transduction, frictional transformation, particle bombardment, or infection.

Representative examples of suitable hosts include bacterial cells such as Streptococcus, Staphylococcus, Escherichia coli, Streptomyces and Bacillus subtilis cells; fungal cells such as yeast cells and Aspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; Animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK 293, and Bowes melanoma cells; and plant cells.

A variety of expression systems can be used. These expression systems include chromosomal, episomal, and virus-derived systems, etc., such as vectors derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses, and combinations of the above elements. Vectors, including baculoviruses, papovaviruses such as SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retroviruses, combination vectors such as genetic elements derived from plasmids and bacteriophages, cosmids and Phagemid carrier. Expression systems may contain control regions that regulate and enable expression. In general, any system or vector suitable for maintaining, propagating or expressing a polynucleotide in a host can be used. Suitable nucleotide sequences can be inserted into expression systems by a variety of known conventional techniques, see, eg, Sambtook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (supra).

In order for the translated protein to be secreted into the lumen of the endoplasmic reticulum, the periplasmic space or the extracellular environment, appropriate secretion signals can be added to the desired polypeptide. These signals can be endogenous to the polypeptide or exogenous secretion signals.

If the CBDAKD01 polypeptide is to be expressed for screening assays, it is generally preferred that the polypeptide be produced on the cell surface. In this case, cells can be harvested prior to use in screening assays. If the CBDAKD01 polypeptide is secreted into the medium, the medium can be recovered to recover and purify the polypeptide; if produced intracellularly, the cells must first be lysed and the polypeptide recovered. CBDAKD01 polypeptides can be recovered and purified from recombinant cell culture by known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography , hydroxyapatite chromatography and lectin chromatography. Most preferably high performance liquid chromatography is used for purification. If the polypeptide is denatured during isolation or purification, known methods of refolding proteins can be used to restore the active conformation. diagnostic test

The present invention also relates to the application of CBDAKD01 polypeptide as a diagnostic reagent. Detection of mutated forms of the CBDAKD01 gene associated with dysfunction can provide a diagnostic tool that can augment or define a diagnosis of a disease or susceptibility to disease due to underexpression, overexpression or aberrant expression of CBDAKD01. Individuals carrying mutations in the CBDAKD01 gene can be detected at the DNA level by a variety of techniques.

Diagnostic nucleic acids can be obtained from cells of a subject, such as blood, urine, saliva, tissue biopsy samples, or autopsy material. Genomic DNA can be used directly for detection, or it can be amplified enzymatically using PCR or other amplification techniques prior to analysis. RNA or cDNA can be used in a similar manner. Deletions and insertions can be detected by changes in the size of the amplified product compared to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled CBDAKD01 nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or differences in melting temperature. DNA sequence differences can also be detected by electrophoretic mobility of DNA fragments on gels with or without denaturing agents or by direct DNA sequencing. See, eg, Myers et al., Science (1985) 230:1242. Sequence changes at specific positions can also be revealed by nuclease protection assays such as RNase and S1 protection or chemical cleavage. See Cotton et al., PNAS USA (1985) 85:4397-4401. In another embodiment, an oligonucleotide probe array (array) containing the CBDAKD01 nucleotide sequence or fragments thereof can be constructed for efficient screening of gene mutations, for example. Array technology methods are well known and widely applicable, and can be used to address a variety of problems in molecular genetics, including gene expression, genetic linkage, and genetic variability. (See e.g. M. Chee et al., Science, Vol. 274, pp610-613 (1996).

Diagnostic assays provide methods for diagnosing or determining susceptibility to hypertension, heart disease, cancer, and kidney disease by detecting mutations in the CBDAKD01 gene using the methods described above.

In addition, hypertension, heart disease, cancer, and kidney disease can be diagnosed by determining an abnormally decreased or increased level of CBDAKD01 polypeptide or CBDAKD01 mRNA in a sample from a subject. Decreased or increased expression can be determined at the RNA level using any method known in the art of nucleotide quantification, such as PCR, RT-PCR, RNase protection, Northern blot and other hybridization methods. Assay techniques that can be used to determine the level of a protein, such as the level of a CBDAKD01 polypeptide, in a sample from a host are well known to those of skill in the art. These assays include radioimmunoassays, competitive binding assays, Western blot analysis, and ELISA assays.

Thus, in another aspect, the present invention relates to a diagnostic kit for determining a disease or susceptibility to a disease, in particular hypertension, heart disease, cancer and kidney disease, said kit comprising: (a) a CBDAKD01 polynucleotide, preferably A nucleotide sequence of sequence 1, or a fragment thereof; (b) a nucleotide sequence complementary to the nucleotides in (a); (c) a CBDAKD01 polypeptide, preferably a polypeptide of sequence 2, or a fragment thereof; (d) a CBDAKD01 polypeptide Antibodies, preferably antibodies to the polypeptide of sequence 2. It should be understood that in such kits, (a), (b), (c) or (d) may comprise a substantial component. chromosome test

The nucleotide sequences of the present invention are also valuable for chromosome identification. The sequence can specifically target and hybridize to a particular location on an individual's chromosome. Mapping of chromosomally associated sequences according to the invention is an important first step in linking these sequences to disease-associated genes. Once a sequence has been mapped to a specific chromosomal location, the sequence's physical location on the chromosome can be linked to genetic map data. These data can be found in, eg, V. McKusick, "Mendelian Inheritance in Humans" (available online from Johns Hopkins University Welch Medical Library). The relationship between genes and diseases that map to the same chromosomal region can be determined by linkage analysis (co-inheritance of physically adjacent genes).

Differences in cDNA or genomic sequences between affected and normal individuals can also be determined. If a mutation is observed in some or all affected individuals but not in normal individuals, the mutation may be the cause of the disease. The CBDAKD01 gene has been mapped to q21.3 on the X chromosome. Antibody

The polypeptides of the present invention or fragments or analogs thereof or cells expressing them can also be used as immunogens to generate antibodies immunospecific for CBDAKD01 polypeptides. "Immunospecific" means in the art that the affinity for the polypeptide of the present invention is significantly greater than the affinity for other related polypeptides in the art.

Antibodies against CBDAKD01 polypeptides can be obtained by administering polypeptides or epitope-containing fragments, analogs or cells to animals, preferably non-human animals, by conventional methods. For the preparation of monoclonal antibodies, any technique that provides antibodies produced by continuous cell line cultures can be used. Examples include hybridoma technology (Kohler, G. and Milstein, C., Nature (1975) 256:495-497), trioma technology, human B cell hybridoma technology (Kozbor et al., Immunology Today (1983 )4:72) and EBV hybridoma technology (Cole et al., "Monoclonal Antibodies and Cancer Therapy", pp77-96, Alan R. Liss, Inc., 1985).

Techniques for the production of single-chain antibodies (US Patent No. 4,946,778) can also be adapted to produce single-chain antibodies to polypeptides of the invention. Transgenic mice or other organisms including other mammals can also be used to express humanized antibodies.

The above-mentioned antibodies can also be used to isolate or identify clones expressing said polypeptides or to purify polypeptides prepared by affinity chromatography.

The antibody against CBDAKD01 polypeptide can also be used to treat hypertension, heart disease, cancer and kidney disease, etc. vaccine

Other aspects of the present invention relate to methods of inducing an immune response in a mammal, comprising immunizing the mammal with a CBDAKD01 polypeptide or a suitable fragment thereof to generate an antibody and/or T cell response, thereby protecting the animal from hypertension, heart disease , cancer and kidney disease. Other aspects of the present invention also relate to a method of inducing an immune response in a mammal, comprising administering a vector that directs the expression of the CBDAKD01 polypeptide in vivo to administer the CBDAKD01 polypeptide, so as to induce an immune response and generate antibodies to protect the animal from disease.

Other aspects of the invention relate to immunogen/vaccine formulations (compositions) that induce an immune response against a CBDAKD01 polypeptide when introduced into a mammalian host, wherein the composition comprises a CBDAKD01 polypeptide or a CBDAKD01 gene. The vaccine formulation may also contain suitable carriers. Because the CBDAKD01 polypeptide can be degraded in the stomach, it is preferably administered through parenteral routes (including subcutaneous, intramuscular, intravenous, intradermal, etc. injection). Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions which may contain antioxidants, buffers, bacteriostats and solutes to render the preparation isotonic with the blood of the recipient; aqueous and non-aqueous sterile Suspensions may contain suspending or thickening agents. The preparation can be in the form of single-dose or multi-dose containers, such as sealed ampoules and vials, and can also be stored in a lyophilized condition by adding a sterile liquid carrier just before use. Vaccine formulations may also contain adjuvant systems that enhance the immunogenicity of the formulation, such as oil-in-water systems or others known in the art. The dosage depends on the specific activity of the vaccine and can be conveniently determined by routine experimentation. screening test

The CBDAKD01 polypeptide of the present invention can be used in a method for screening compounds that activate (agonist) or inhibit (antagonist or inhibitor) the activation of the CBDAKD01 polypeptide of the present invention. The polypeptides of the invention can therefore also be used to evaluate and identify agonists or antagonists from cells, cell-free preparations, chemical libraries and mixtures of natural products. These agonists or antagonists may be natural or modified substrates, ligands, receptors, enzymes, etc. of the polypeptides of the present invention; or structural or functional mimics of the polypeptides of the present invention. See Coligan et al., Current Protocol in Immunology 1(2): Chapter 5 (1991).

The CBDAKD01 polypeptide has various physiological functions, including various pathological effects. Therefore, it is necessary to find compounds and drugs that stimulate CBDAKD01 polypeptide or inhibit the function of CBDAKD01 polypeptide. In general, agonists can be used for therapeutic and preventive purposes in diseases such as hypertension, heart disease, cancer and kidney disease. Antagonists can be used for a variety of therapeutic and preventive purposes in diseases such as hypertension, heart disease, cancer and kidney disease.

Generally, these screening methods involve the use of suitable cells that express a CBDAKD01 polypeptide or are responsive to a CBDAKD01 polypeptide of the invention. These cells include cells from mammals, yeast, Drosophila or E. coli. Cells expressing the CBDAKD01 polypeptide (or containing cell membranes expressing the polypeptide) or cells responding to the CBDAKD01 polypeptide are contacted with the test compound, and the binding or stimulation or inhibition of the functional response is observed. The ability to compare the CBDAKD01 activity of cells exposed to the candidate compound with the same cells not exposed to the candidate compound.

The assay can simply test the binding of the candidate compound, where adhesion to cells bearing the CBDAKD01 polypeptide is detected by a label that binds directly or indirectly to the candidate compound, or by an assay involving competition with a labeled competitor. Also, these assays can test whether a candidate compound produces a signal for activation of the CBDAKD01 polypeptide, using a detection system suitable for cells bearing the CBDAKD01 polypeptide. Activation inhibitors are typically tested in the presence of a known agonist to observe the effect of the presence of the candidate compound on the activation of the agonist.

In addition, the test may simply include the following steps: mixing the candidate compound with a solution containing the CBDAKD01 polypeptide to form a mixture, measuring the activity of CBDAKD01 in the mixture, and comparing the CBDAKD01 activity of the mixture with a standard product.

CBDAKD01 cDNA, protein and protein antibodies can also be used to design assays to detect the effect of added compounds on CBDAKD01 mRNA and protein production in cells. For example, ELISAs can be designed to measure levels of secreted or cell-bound CBDAKD01 protein using monoclonal and polyclonal antibodies by standard methods known in the art, which can be used to search for cells or tissues that inhibit or Substances that increase the production of CBDAKD01 (may also be referred to as antagonists or agonists, respectively).

The CBDAKD01 protein can be used to identify the presence of membrane bound or soluble receptors using standard receptor binding techniques known in the art. These include, but are not limited to, ligand binding and cross-linking assays, in which CBDAKD01 is radiolabeled (125I), chemically modified (e.g., biotinylated), or fused to a peptide sequence suitable for detection or purification, with a putative receptor source ( cells, cell membranes, cell supernatants, tissue extracts, body fluids). Other methods include biophysical techniques such as surface plasmon resonance and spectroscopy. In addition to being used to purify and clone receptors, these binding assays can also be used to identify CBDAKD01 agonists and antagonists that compete with the binding of CBDAKD01 to existing receptors. Standard methods for performing screening assays are well known in the art.

Examples of possible CBDAKD01 polypeptide antagonists include antibodies to CBDAKD01 polypeptides, or in some cases oligonucleotides or proteins closely related to ligands, substrates, receptors, enzymes, etc. of CBDAKD01 polypeptides, such as ligands, Fragments of substrates, receptors, enzymes, etc.; or small molecules that bind to the polypeptide of the invention but do not elicit a response, thereby inhibiting the activity of the polypeptide.

Therefore, another aspect of the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. of CBDAKD01 polypeptides; or compounds that reduce or increase the production of CBDAKD01 polypeptides. The kit includes: (a) CBDAKD01 polypeptide, preferably the polypeptide of sequence 2; (b) recombinant cells expressing the polypeptide of preferred sequence 2 of CBDAKD01 polypeptide; (c) cell membranes expressing the polypeptide of preferred sequence 2 of CBDAKD01 polypeptide; (d) CBDAKD01 polypeptide Antibodies, preferably antibodies to the polypeptide of sequence 2. It should be understood that in such kits, (a), (b), (c) or (d) may comprise an essential ingredient. Prevention and Treatment

The present invention provides a treatment method for diseases related to high or low activity of CBDAKD01 polypeptide, such as hypertension, heart disease, cancer and kidney disease.

If the activity of CBDAKD01 polypeptide is too high, there are several ways. One method comprises administering to a subject an inhibitor compound (antagonist) as described above and a pharmaceutically acceptable carrier in an amount capable of inhibiting the function of the CBDAKD01 polypeptide, for example by blocking the binding of ligands, substrates, receptors, enzymes, etc. , or by inhibiting the second signal, thereby alleviating abnormal symptoms. In another method, a soluble form of the CBDAKD01 polypeptide is administered that is capable of competing with the endogenous CBDAKD01 polypeptide for binding to ligands, substrates, enzymes, receptors, and the like. Typical examples of such competitors include fragments of CBDAKD01 polypeptides.

In another approach, expression of the gene encoding the endogenous CBDAKD01 polypeptide can be inhibited using expression blocking techniques. Known techniques of this kind include the use of antisense sequences, either generated internally or administered separately. See, eg, O'Connor, J Neurochem (1991) 56:560, Oligodeoxynucleotides for Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988). Alternatively oligonucleotides that form a triple helix with the gene can be used. See, eg, Lee, et al., Nucleic Acid Res (1979) 6:3073; Cooney et al., Science (1988) 241:456; Dervan et al., Scicence (1991) 251:1360. These oligomers can be administered or expressed in vivo.

In order to treat CBDAKD01 and abnormal symptoms of insufficient expression of its activity, several methods are available. One method includes administering to a subject a therapeutically effective amount of a compound that activates CBDAKD01 polypeptide, that is, the aforementioned agonist, in combination with a pharmaceutically acceptable carrier, thereby alleviating abnormal symptoms. Alternatively, gene therapy can be used to achieve endogenous production of CBDAKD01 in relevant cells of the subject. For example, polynucleotides of the invention can be expressed in replication-deficient retroviral vectors engineered as described above. The retroviral expression construct is then isolated and introduced into packaging cells transduced with a retroviral plasmid vector containing RNA encoding the polypeptide of the invention so that the packaging cells can produce infectious virions containing the gene of interest. These producer cells can be administered to a subject to engineer the cells in vivo and express the polypeptide in vivo. For a discussion of gene therapy see: Chapter 20, "Gene Therapy and Other Molecular Genetics-Based Therapeutics (and references therein), Human Molecular Genetics, T Strachan and A P Read, BIOS Scientific Publishers Ltd ( 1996). Another method is to administer a therapeutically effective amount of CBDAKD01 polypeptide in combination with a suitable pharmaceutical carrier. Formulation and administration

Peptides such as soluble forms of CBDAKD01 polypeptides and agonists and antagonists of peptides or small molecules can be formulated in combination with suitable pharmaceutical carriers. These formulations include a therapeutically effective amount of a polypeptide or compound and a pharmaceutically acceptable carrier or excipient. These carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration, as is well known to those skilled in the art. The invention also relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the above-described composition components of the invention.

The polypeptides and other compounds of the invention can be used alone, or in combination with other compounds, such as therapeutic compounds.

Preferred forms of systemic administration of the pharmaceutical compositions include injection, typically intravenous injection. Other routes of injection such as subcutaneous, intramuscular, or intraperitoneal routes may also be used. Other methods of systemic administration include transmucosal, transdermal administration using penetrants such as cholate or fusidic acid or other surfactants. Additionally, oral administration may also be possible if formulated as enteral or capsule form. The compounds may also be administered topically and/or topically in the form of ointments, pastes, gels and the like.

The desired dosage range will depend on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's symptoms and the judgment of the attending physician. A suitable dosage range is 0.1 to 100 μg/kg. Given the variety of compounds available and the different effects of different routes of administration, wide variations in the required dosage are to be expected. For example, oral administration is expected to require larger doses than intravenous administration. Variations in these dosage levels can be adjusted using standard experimentation for optimization, as is well known in the art.

Therapeutic polypeptides can also be produced endogenously in a subject, a form of treatment commonly referred to as "gene therapy." Thus, for example, cells from a subject can be engineered with polynucleotides, such as DNA or RNA encoding ex vivo polypeptides, using retroviral plasmid vectors. These cells are then introduced into the subject.

All publications, including but not limited to patents and patent applications, cited in this specification are hereby incorporated by reference in their entirety as if each individual publication were individually and individually indicated to be incorporated by reference.

sequence listing

(1) General information (ⅰ) Applicants: Zhang Qinghua, Shen Yu, Mao Mao, Gu Baiwei (ⅱ) Invention name: Human angiotensin II/vasopressin receptor (AII/AVP)-like gene

Due to (CBDAKD01) (ⅳ) serial number: 2 (ⅳ) mailing address:

(A) Contact: Ratner & Prestia

(B) Street: P.O. Box 980

(C) City: Valley Forge

(D) State: PA

(E) Country: USA

(F) Zip code: 19482 (ⅴ) Computer readable form:

(A) Media type: floppy disk

(B) Computer: IBM Compatible

(C) Operating system: DOS

(D) Software: FastSEQ for Windows Version 2.0 (ⅵ) Current application data:

(A) Application number:

(B) Filing date:

(C) Classification: (ⅶ) Prior application data:

(A) Application number:

(B) Filing Date: (ⅷ) Lawyer/Representative Information:

(A) Name: Prestia, Paul F

(B) Registration number: 23,031

(C) Reference/Docket No.: GP-70382 (ⅸ) Telecommunication Information:

(A) Tel: 610/407-0700

(B) Fax: 610/407-0701

(C) Telex: 846169

(2) Sequence 1 information: (i) Sequence features:

(A) Length: 2218 base pairs

(B) type: nucleic acid

(C) Chain type: single chain

(D)拓扑结构：线性(ⅱ)分子类型：cDNA(ⅹⅰ)序列表述：序列1：CAGGGCAGCC TTCAGTCTGA TTCAGGAGAA CGAGGTCCTC TTCACCATGT GCTTCATCCC       60CCTGGTCTGC TGGATCGTGT GCACTGGACT GAAACAGCAG ATGGAGAGTG GCAAGAGCCT      120TGCCCAGACA TCCAAGACCT CCACCGCGGT GTACGTCTTC TTCCTTTCCA GTTTGCTGCA      180GCCCCGGGGA GGGAGCCAGG AGCACGGCCT CTGCGCCCAC CTCTGGGGGC TCTGCTCTTT      240GGCTGCAGAT GGAATCTGGA ACCAGAAAAT CCTGTTTGAA GAGTCCGACC TCAGGAATCA      300TGGACTGCAG AAGGCGGATG TGTCTGCTTT CCTGAGGATG AACCTGTTCC AAAAGGAAGT      360GGACTGCGAG AAGTTCTACA GCTTCATCCA CATGACTTTC CAGGAGTTCT TTGCCGCCAT      420GTACTACCTG CTGGAAGAGG AAAAGGAAGG AAGGACGAAC GTTCCAGGGA GTCGTTTGAA      480GCTTCCCAGC CGAGACGTGA CAGTCCTTCT GGAAAACTAT GGCAAATTCG AAAAGGGGTA      540TTTGATTTTT GTTGTACGTT TCCTCTTTGG CCTGGTAAAC CAGGAGAGGA CCTCCTACTT      600GGAGAAGAAA TTAAGTTGCA TGATCTCTCA GCAAATCAGG CTGGAGCTGC TGAAATGGAT      660TGAAGTGAAA GCCAA GCTA AAAAGCTGCA TGATCAGCCC AGCCAGCTGG AATTGTTCTA      720CTGTTTGTAC GAGATGCAGG AGGAGGACTT CGTGCAAAGG GCCATGGACT ATTTCCCCAA      780GATTGAGATC AATCTCTCCA CCAGAATGGA CCACATGGTT TCCTCCTTTT GCATTGAGAA      840CTGTCATCGG GTGGAGTCAC TGTCCCTGGG GTTTCTCCAT AACATGCCCA AGGAGGAAGA      900GGAGGAGGAA AAGGAAGGCC GACACCTTGA TATGGTGCAG TGTGTCCTCC CAAGCTCCTC      960TCATGCTGCC TGTTCTCATG GGTTGGGGCG CTGTGGCCTC TCCCATGAGT GCTGCTTCGA     1020CATCTCCTTG GTCCTCAGCA GCAACCAGAA GCTGGTGGAG CTGGACCTGA GTGACAACGC     1080CCTCGGTGAC TTCGGAATCA GACTTCTGTG TGTGGGACTG AAGCACCTGT TGTGCAATCT     1140GAAGAAGCTC TGGTTGGTGA ATTCTGCCTT ACGTCAGTCT GTTGTTCAGC TTTGTCCTCG     1200GTACTCAGCA CTAATCAGAA TCTCACGCAC CTTTACTGCG AGGCAACACT CTCGGAGACA 1260AGGGATCAAA CTACTCTGTG AGGGACTCTT GCACCCCGAC TGcAAGCTTC AGGTGTTGGA     1320ATTAGACAAC TGCAACCTCA CGTCACACTG CTGCTGGGAT CTTTCCACAC TTCTGACCTC      1380CAGCCAGAGC CTGCGAAAGC TGAGCCTGGG CAACAATGAC CTGGGCGACC TGGGGGTCAT      1440GATGTTCTGT GAAGTGCTGA AACAGCAGAG CTGCCTCCTG CAGAACCTGG GGTTGTCTGA      1500AATGTATTTC AATTATGAGA CAAAAAGTGC GTTAGAAACA CTTCAAGAAG AAAAGCCTGA      1560GCTGACCGTC GTCTTTGAGC CTTCTTGGTA GGAGTGGAAA CGGGGCTGCC AGACGCCAGT      1620GTTCTCCGGT CCCTCCAGCT GGGGGCCCTC AGGTGGAGAG AGCTGCGATC CATCCAGGCC      1680AAGACCACAG CTCTGTGATC CTTCCGGTGG AGTGTCGGAG AAGAGAGCTT GCCGACGATG      1740CCTTCCTGTG CAGAGCTTGG GCATCTCCTT TACGCCAGGG TGAGGAAGAC ACCAGGACAA      1800TGACAGCATC GGGTGTTGTT GTCATCACAG CGCCTCAGTT AGAGGATGTT CCTCTGGTGA      1860CCTCATGTAA TTAGCTCATT CAATAAAGCA CTTTCTTTAT TTTTCTCTTC TCTGTCTAAC      1920CTTCTTTTTC CTATCTTTTT TTCTTCTTTG TTCTGTTTAC TTTTGCTCAT ATCATCATTC      1980CCGCTATCTT TCTATTAACT GACCATAACA CAGAACTAGT TGACTATATA TTATGTTGAA      2040ATTTTATGGC AGCTATTTAT TTATTTAAAT TTTTTGTAAT AGTTTTGTTT TCTAATAAGA      2100AAAATCCATG CTTTTTGTAG CTGGTTGAAA ATTCAGGAAT ATGTAAAACT TTTTGGTATT      2160TAATTAAATT GATTCCTTTT CTTAATTTTA AAAAAAAAAA AAAAAAAAAA AAAAAAAA        2218

(2) Sequence 2 information: (i) Sequence features:

(A) Length: 514 amino acids

(B) Type: amino acid

(C) Chain type: single chain

(D) Topology: Linear (Ⅱ) Molecular type: Protein (ⅹ Ⅰ) sequence expression: Sequence 2: Met Cys PHE Ile Pro Leu Val Cys TRP Ile Val Cys THR GLY Leu LYS1 5 10 15GLN Met GLY LYS Serou Ala Gln Thr Ser Lys Thr Ser20                  25                  30Thr Ala Val Tyr Val Phe Phe Leu Ser Ser Leu Leu Gln Pro Arg Gly35                  40                  45Gly Ser Gln Glu His Gly Leu Cys Ala His Leu Trp Gly Leu Cys Ser50                  55                  60Leu Ala Ala Asp Gly Ile Trp Asn Gln Lys Ile Leu Phe Glu Glu Ser65                  70                  75                  80Asp Leu Arg Asn His Gly Leu Gln Lys Ala Asp Val Ser Ala Phe Leu85                  90                  95Arg Met Asn Leu Phe Gln Lys Glu Val Asp Cys Glu Lys Phe Tyr Ser100                 105                 110Phe Ile His Met Thr Phe Gln Glu Phe Phe Ala Ala Met Tyr Tyr Leu        115                 120                 125Leu Glu Glu Glu Lys Glu Gly Arg Thr Asn Val Pro Gly Ser Arg Leu130                 135                 140Lys Leu Pro Ser Arg Asp Val Thr Val Leu Leu Glu Asn Tyr Gly Lys145                 150                 155                 160Phe Glu Lys Gly Tyr Leu Ile Phe Val Val Arg Phe Leu Phe Gly Leu165                 170                 175 Val Asn Gln Glu Arg Thr Ser Tyr Leu Glu Lys Lys Leu Ser Cys Met180                 185                 190Ile Ser Gln Gln Ile Arg Leu Glu Leu Leu Lys Trp Ile Glu Val Lys195                 200                 205Ala Lys Ala Lys Lys Leu His Asp Gln Pro Ser Gln Leu Glu Leu Phe210                 215                 220Tyr Cys Leu Tyr Glu Met Gln Glu Glu Asp Phe Val Gln Arg Ala Met225                 230                 235                 240Asp Tyr Phe Pro Lys Ile Glu Ile Asn Leu Ser Thr Arg Met Asp His245 250                 255Met Val Ser Ser Phe Cys Ile Glu Asn Cys His Arg Val Glu Ser Leu260                 265                 270Ser Leu Gly Phe Leu His Asn Met Pro Lys Glu Glu Glu Glu Glu Glu275                 280                 285Lys Glu Gly Arg His Leu Asp Met Val Gln Cys Val Leu Pro Ser Ser290                 295                 300Ser His Ala Ala Cys Ser His Gly Leu Gly Arg Cys Gly Leu Ser His305                 310                 315                 320Glu Cys Cys Phe Asp Ile Ser Leu Val Leu Ser Ser Asn Gln Lys Leu325                 330                 335Val Glu Leu Asp Leu Ser Asp Asn Ala Leu Gly Asp Phe Gly Ile Arg340                 345                 350Leu Leu Cys Val Gly Leu Lys His Leu Leu Cys Asn Leu Lys Lys Leu355                 360                 365Trp Leu Val Asn Ser Ala Leu Arg Gln Ser Val Val Gln Leu Cys Pro370                 375                 380Arg Tyr Ser Ala Leu Ile Arg Ile Ser Arg Thr Phe Thr A la Arg Gln385                 390                 395                  400His Ser Arg Arg Gln Gly Ile Lys Leu Leu Cys Glu Gly Leu Leu His405                 410                 415Pro Asp Cys Lys Leu Gln Val Leu Glu Leu Asp Asn Cys Asn Leu Thr420                 425                 430Ser His Cys Cys Trp Asp Leu Ser Thr Leu Leu Thr Ser Ser Gln Ser435                 440                 445Leu Arg Lys Leu Ser Leu Gly Asn Asn Asp Leu Gly Asp Leu Gly Val450                 455                 460Met Met Phe Cys Glu Val Leu Lys Gln Gln Ser Cys Leu Leu Gln Asn465                 470                 475                 480Leu Gly Leu Ser Glu Met Tyr PHE Asn Tyr Glu Thr Lys Ser Ala Leu485 495GLU Thr Leu Gln Glu Lys Pro Glu Leu ThR Val PHE GLU500 510SER TRP

Claims (19)

1. isolating polynucleotide contain identity with the nucleotide sequence total length of the CBDAKD01 polypeptide of encoding sequence 2 and are at least 80% nucleotide sequence; The complementary nucleotide sequence of perhaps described separation polynucleotide.

2. the polynucleotide of claim 1, wherein said polynucleotide comprise the nucleotide sequence of sequence 1 of the CBDAKD01 polypeptide of encoding sequence 2.

3. the polynucleotide of claim 1, wherein said polynucleotide comprise that the identity with the nucleotide sequence total length of sequence 1 is at least 80% nucleotide sequence.

4. the polynucleotide of claim 3, it is the polynucleotide of sequence 1.

5. the polynucleotide of claim 1, it is DNA or RNA.

6. contain a kind of DNA or RNA molecule of expression system, wherein said expression system can be produced the CBDAKD01 polypeptide in compatible host cell, and the identity of the polypeptide of its aminoacid sequence and sequence 2 is at least 80%.

7. the host cell that contains the expression system of claim 6.

8. produce the method for CBDAKD01 polypeptide, be included in the host who cultivates claim 7 under the condition that can produce described polypeptide, by reclaiming polypeptide in the culture.

9. preparation produces the method for the cell of CBDAKD01 polypeptide, comprises that the expression system with claim 6 transforms or transfection host cell, makes host cell can produce the CBDAKD01 polypeptide under suitable culture condition.

10.CBDAKD01 polypeptide contains identity with the aminoacid sequence total length of sequence 2 and is at least 80% aminoacid sequence.

11. the polypeptide of claim 10, it comprises the aminoacid sequence of sequence 2.

12. the immunologic opsonin antibody of the CBDAKD01 polypeptide of claim 10.

13. the experimenter's who treats the activity of the CBDAKD01 polypeptide that needs increase claim 10 or express method comprises:

(a) to the agonist of the described polypeptide of experimenter's drug treatment significant quantity; And/or

(b) provide isolating polynucleotide to the experimenter, the identity of the coding nucleotide sequence total length of the CBDAKD01 polypeptide of its nucleotide sequence and sequence 2 is at least 80%; Perhaps its nucleotide sequence and above-mentioned nucleotide sequence complementation, the form of polynucleotide can realize the expression in vivo of described polypeptide active.

14. the experimenter's who treats the activity of the CBDAKD01 polypeptide that needs inhibition claim 10 or express method comprises:

(a) to the antagonist of the described polypeptide of experimenter's drug treatment significant quantity; And/or

(b) suppress the nucleic acid molecule that the coding nucleotide sequence of described polypeptide is expressed to experimenter's administration; And/or

(c) compete the polypeptide of its part, substrate or acceptor to experimenter's drug treatment significant quantity with described polypeptide.

15. with the method for the susceptibility of the CBDAKD01 polypeptide expression of claim 10 or active diseases associated or disease, comprising among the diagnosis experimenter:

(a) determine whether there is sudden change in the coding nucleotide sequence of CBDAKD01 polypeptide described in described experimenter's genome; And/or

(b) analyze from whether having CBDAKD01 polypeptide expression or its expression amount in experimenter's the sample.

16. identify the method for the compound of the CBDAKD01 polypeptide that suppresses (antagonism) or activation claim 10, comprising:

(a) with candidate compound and the cell (or cytolemma of expression CBDAKD01 polypeptide) of expression CBDAKD01 polypeptide or the cells contacting that the CBDAKD01 polypeptide is responded; With

(b) stimulation or the inhibition of observation combination or functional response; Or the cell of more contacted candidate compound (or cytolemma) and the ability of the isocellular CBDAKD01 polypeptide active of contacted candidate compound not.

17. the agonist that the method for claim 16 is identified.

18. the antagonist that the method for claim 16 is identified.

19. its cytolemma of the recombinant host cell of the method for claim 9 preparation or expression CBDAKD01 polypeptide.