WO2001070983A1 - A novel polypeptide, a human prostate specific membrane antibody proteine 9 and the polynucleotide encoding the polypeptide - Google Patents

Abstract

The present invention discloses a novel polypeptide, a human prostate specific membrane antibody protein 9, the polynucleotide encoding the polypeptide and the method for producing the polypeptide by DNA recombinant technology. The invention also discloses the uses of the polypeptide in methods for treating various diseases, such as carcinoma of prostate, begnin tumor of prostate, other tumour, folic acid metabolic deficient illness, neural mental illness, inflammation, immunological disease, hemopathy, development disorder, HIV infection, etc. The invention also discloses the agonists against the polypeptide and the therapeutic action thereof. The invention also discloses the uses of the polynucleotide encoding the novel human prostate specific membrane antibody protein 9.

Description

 A new polypeptide-human prostate-specific membrane antibody protein 9 and a polynucleotide encoding the polypeptide TECHNICAL FIELD

 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide, a human prostate-specific membrane antibody protein 9 and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides. Background technique

 Prostate-specific membrane antibody protein is a type II transmembrane protein that is expressed in prostate and non-prostate tissues. Studies have found that prostate-specific membrane antibody protein is a transmembrane glycoprotein, which has a high expression level in the prostate-related tissues, including benign secretory acinar epithelium, prostate epithelial tumors, and prostate cancer cells. The gene encoding this protein is located on the short arm of human chromosome 11. It has both folate hydrolase and neuropeptidase activity in the body, and is involved in regulating various important metabolic processes. The mutation or abnormal expression of this protein may be closely related to the occurrence of diseases such as tumors and cancers in various related tissues in the body.

 People have cloned a variety of prostate-specific membrane antibody proteins from various organisms. In 1999, Sam et al. Cloned a prostate-specific membrane antibody protein from humans and found that the protein is not only related to the prostate. Tissues have a higher expression level, and also have higher expression levels in some solid tumors, tumor-associated neovascularization and other cells. It can be known that this protein has a high expression level in some related tumor cells, and it acts as an antibody protein in the body to inhibit the development of related tumors. The mutation or abnormal expression of this protein may be closely related to the occurrence of malignant diseases such as tumors and cancer in related tissues [Sam S. Chang, Deni se S. O'Keefe et al., 1999, CI inical Cancer Resea rch, 2: 2674-2681].

 From the above, it is known that prostate-specific membrane antibody protein, as a transmembrane glycoprotein, regulates the occurrence of diseases such as tumors and cancers in related tissues. The protein is highly expressed not only in the prostate and some related tissues, but also in benign and malignant prostate tumors, solid tumors, tumor endothelial cells, non-prostate tumors and other cells. Mutation or abnormal expression of this protein will often lead to abnormal proliferation of the above-mentioned various cells, which is closely related to the occurrence of tumors and cancers in various related tissues in the body. It can also be used to diagnose and treat various diseases mentioned above.

Gene chip analysis revealed that in the thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, arsenic-stimulated L02 In cell lines and prostate tissues, the expression profiles of the polypeptides of the present invention and The expression profiles of human prostate-specific membrane antibody proteins are very similar, so their functions may be similar. The invention is named human prostate specific membrane antibody protein 9.

 As mentioned above, the human prostate-specific membrane antibody protein 9 protein plays an important role in regulating important functions of the body such as cell division and embryonic development, and it is believed that a large number of proteins are involved in these regulatory processes, so there has been a need to identify more participation in the field. These processes are specific for the human prostate-specific membrane antibody protein 9 protein, especially the amino acid sequence of this protein. Isolation of the novel prostate-specific membrane antibody protein 9 protein encoding gene also provides the basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so it is important to isolate its coding DNA. Disclosure of invention

 It is an object of the present invention to provide isolated novel polypeptides-human prostate-specific membrane antibody protein 9 and fragments, analogs and derivatives thereof.

 Another object of the invention is to provide a polynucleotide encoding the polypeptide.

 Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human prostate-specific membrane antibody protein 9.

 Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human prostate-specific membrane antibody protein 9.

 Another object of the present invention is to provide a method for producing human prostate-specific membrane antibody protein 9.

 Another object of the present invention is to provide an antibody against the polypeptide of the present invention-human prostate-specific membrane antibody protein 9.

 Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors against the human prostate-specific membrane antibody protein 9 of the polypeptide of the present invention.

 Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities of human prostate-specific membrane antibody protein 9.

 The present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.

 The invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:

 (a) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID No. 2;

(b) a polynucleotide complementary to polynucleotide (a); (c) A polynucleotide having at least 70% identity to a polynucleotide sequence of (a) or (b).

 More preferably, the sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 1477-1722 in SEQ ID NO: 1; and (b) having a sequence of 1-3321 in SEQ ID NO: 1 Sequence of bits.

 The present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.

 The invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.

 The invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of human prostate specific membrane antibody protein 9 protein, which comprises utilizing the polypeptide of the invention. The invention also relates to compounds obtained by this method.

 The present invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of a human prostate-specific membrane antibody protein 9 protein in vitro, which comprises detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or Detection of the amount or biological activity of a polypeptide of the invention in a biological sample.

 The invention also relates to a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.

 The present invention also relates to the preparation of the polypeptide and / or polynucleotide of the present invention for the treatment of prostate cancer, benign prostate tumors, other tumors, folic acid deficiency disorders, neuropsychiatric diseases, inflammation, immune diseases, blood diseases, development disorders Disease, HIV infection or other diseases caused by abnormal expression of human prostate-specific membrane antibody protein-9.

 Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein.

 The following terms used in this specification and claims have the following meanings unless specifically stated: "Nucleic acid sequence" refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genome or a synthetic DM or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand. Similarly, the term "amino acid sequence" refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof. When the "amino acid sequence" in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide" or "protein" does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .

A protein or polynucleotide "variant" refers to an amino acid sequence having one or more amino acids or nucleotide changes, or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes, in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of Leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.

 "Deletion" refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.

 "Insertion" or "addition" refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule. "Replacement" refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.

 "Biological activity" refers to a protein that has the structure, regulation, or biochemical function of a natural molecule. Similarly, the term "immunologically active" refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.

 An "agonist" refers to a molecule that, when bound to a human prostate-specific membrane antibody protein 9, can cause changes in the protein and thereby regulate the activity of the protein. An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to human prostate-specific membrane antibody protein 9.

 An "antagonist" or "inhibitor" refers to a molecule that can block or regulate the biological or immunological activity of human prostate-specific membrane antibody protein 9 when combined with human prostate-specific membrane antibody protein 9. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to human prostate-specific membrane antibody protein 9.

 "Regulation" refers to a change in the function of human prostate-specific membrane antibody protein 9, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological properties, functions, or immunity of human prostate-specific membrane antibody protein 9. Change of nature.

 By "substantially pure" is meant substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify human prostate-specific membrane antibody protein 9 using standard protein purification techniques. Substantially pure human prostate-specific membrane antibody protein 9 produces a single main band on a non-reducing polyacrylamide gel. The purity of human prostate-specific membrane antibody protein 9 polypeptide can be analyzed by amino acid sequence.

 "Complementary" or "complementary" refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature. For example, the sequence "C-T-G-A" can be combined with the complementary sequence "G-A-C-T". The complementarity between two single-stranded molecules may be partial or complete. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

"Homology" refers to the degree of complementarity and can be partially homologous or completely homologous. "Partial homology" refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. Inhibition of such hybridization can be detected by performing hybridization (Southern blotting or Nor thern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean strict Conditions with reduced sex allow non-specific binding because conditions with reduced stringency require that the two sequences bind to each other as a specific or selective interaction.

"Percent identity" refers to the percentage of sequences that are identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences based on different methods such as the Clus ter method (Higg ins, DG and PM Sharp (1988) Gene 73: 237-244). _{0 The} Clus ter method compares each pair by checking the distance between all pairs. Group sequences are arranged in clusters. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence X 100 The number of residues in sequence A-the number of spacer residues in sequence A The number of spacer residues in a sequence B can also be determined by Cluster method or using methods known in the art such as Jotun He in (% in Hez J., (1990) Methods in emzumology 183: 625 -645) ₀

"Similarity" refers to the degree to which the amino acid residues at the corresponding positions are identical or conservatively substituted when the amino acid sequences are aligned. Amino acids used for conservative substitutions, for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.

 "Antisense" refers to a nucleotide sequence that is complementary to a particular DM or RM sequence. "Antisense strand" refers to a nucleic acid strand that is complementary to a "sense strand."

 "Derivative" refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.

"Antibody" refers to a complete antibody molecule and its fragments, such as Fa,? (') ₂ and? ^ It can specifically bind to the human prostate-specific membrane antibody protein 9 epitope.

 A "humanized antibody" refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.

The term "isolated" refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally). For example, a naturally occurring polynucleotide or polypeptide exists in a living animal. It is not isolated, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist with it in the natural system. Such a polynucleotide may be part of a certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.

 As used herein, "isolated" refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances in the natural state .

 As used herein, "isolated human prostate-specific membrane antibody protein 9" refers to human prostate-specific membrane antibody protein 9 which is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify human prostate-specific membrane antibody protein 9 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human prostate-specific membrane antibody protein 9 polypeptide can be analyzed by amino acid sequence.

 The present invention provides a new polypeptide, a human prostate-specific membrane antibody protein 9, which basically consists of the amino acid sequence shown in SEQ ID NO: 2. The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide. The polypeptides of the present invention can be naturally purified products, or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.

The invention also includes fragments, derivatives, and analogs of human prostate-specific membrane antibody protein 9. As used in the present invention, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially maintains the same biological function or activity of the human prostate-specific membrane antibody protein 9 of the present invention. A fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution The amino acid may or may not be encoded by the genetic code or (Π) such that a group on one or more amino acid residues is substituted by another group to include a substituent; or (Π Ι) like this A type in which a mature polypeptide is fused to another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol); or (IV) a type of polypeptide sequence in which an additional amino acid sequence is fused into a mature polypeptide ( Such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence) As explained herein, such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art. The present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. The polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1. The polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence with a total length of 3321 bases, and its open reading frame 1477-1722 encodes 81 amino acids. According to the comparison of gene chip expression profiles, it was found that this polypeptide has a similar expression profile to human prostate-specific membrane antibody protein, and it can be deduced that the human prostate-specific membrane antibody protein 9 has similar functions to human prostate-specific membrane antibody protein.

 The polynucleotide of the present invention may be in the form of DNA or RM. DNA forms include cDNA, genomic DNA, or synthetic DM. DNA can be single-stranded or double-stranded. DM can be a coding chain or a non-coding chain. The coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant. As used herein, a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but having a sequence different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.

 The polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.

 The term "polynucleotide encoding a polypeptide" refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.

 'The present invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention. Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .

The present invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50 »/, and preferably 70% identity between the two sequences). The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "strict conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 6 (TC; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% F i co ll, 42 ° C, etc .; or (3) only between the two sequences The hybridization occurs only when the identity between them is at least 95%, and more preferably 97%. Moreover, the polypeptide encoded by the hybridizable polynucleotide has the same biological function as the mature polypeptide shown in SEQ ID NO: 2 and active. The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used herein, a "nucleic acid fragment" contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nucleotides. Nucleotides or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding human prostate-specific membrane antibody protein 9.

 The polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity. The specific polynucleotide sequence encoding the human prostate-specific membrane antibody protein 9 of the present invention can be obtained by various methods. For example, polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.

 The DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DM of the polypeptide.

 Of the methods mentioned above, genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences. The standard method for isolating the cDM of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for mRNA extraction. Kits are also commercially available (Qiagene). And the construction of cDNA libraries is also a common method (Sambrook, et al., Molecule Cloning, A Labora tory Manua, Collspring Harbor Laboratory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.

 The genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or loss of marker gene function; (3) determination of the level of the human prostate-specific membrane antibody protein 9 transcript; (4) Detecting the protein product of gene expression by immunological technology or measuring biological activity. The above methods can be used alone or in combination.

 In the method (1), the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides. In addition, the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides. The probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).

In the (4) method, the protein product of human prostate specific membrane antibody protein 9 gene expression is detected Available immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).

 A method using DNA technology to amplify DNA / RNA (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. In particular, when it is difficult to obtain full-length CDM from a library, the RACE method (RACE-rapid amplification of cDNA ends) can be preferably used, and the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein Select and synthesize using conventional methods. The amplified DM / RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.

 The polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDM sequence of multiple clones in order to splice into a full-length cDNA sequence.

 The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using a human prostate-specific membrane antibody protein 9 coding sequence, and the recombinant technology to produce the polypeptide of the present invention Methods.

 In the present invention, a polynucleotide sequence encoding a human prostate-specific membrane antibody protein 9 may be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention. The term "vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors known in the art. Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized within the host's body, any plasmid and vector can be used to construct a recombinant expression vector. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.

Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human prostate-specific membrane antibody protein 9 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989). The DM sequence can be operably linked to an appropriate promoter in the expression vector to guide the synthesis of raRM. Representative examples of these promoters are: the lac or trp promoter of E. coli; the PL promoter of lambda phage; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early Phase and late SV40 promoters, retroviral LTRs and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs at a later stage of the origin of replication, polyoma enhancers and adenovirus enhancers at the late side of the origin of replication, and the like.

 In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture. Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

 Those of ordinary skill in the art will know how to select appropriate vector / transcription control elements (such as promoters, enhancers, etc.) and selectable marker genes.

 In the present invention, a polynucleotide encoding a human prostate-specific membrane antibody protein 9 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host cell containing the polynucleotide or the recombinant vector. . The term "host cell" refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf 9; animal cells such as CH0, COS or Bowes melanoma cells.

Transformation of a host cell with a DNA sequence according to the present invention or a recombinant vector containing the DM sequence can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DM can be harvested after the exponential growth phase and treated with the CaCl ₂ method. The steps used are well known in the art. Alternatively, M _g Cl ₂ is used. If necessary, transformation can also be performed by electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.

 The polynucleotide sequence of the present invention can be used to express or produce recombinant human prostate-specific membrane antibody protein 9 by conventional recombinant DNA technology (Science, 1984; 224: 1431). Generally, the following steps are taken:

 (1) using the polynucleotide (or variant) encoding the human prostate-specific membrane antibody protein 9 of the present invention, or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide;

 (2) culturing host cells in a suitable medium;

 (3) Isolate and purify protein from culture medium or cells. '

In step (2), depending on the host cell used, the medium used in the culture may be selected from various Conventional medium. Culture is performed under conditions suitable for host 'cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

 In step (3), the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If desired, recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods. Brief description of the drawings

 The following drawings are used to illustrate specific embodiments of the invention, but not to limit the scope of the invention as defined by the claims.

 Fig. 1 is a comparison diagram of gene chip expression profiles of human prostate-specific membrane antibody protein 9 and human prostate-specific membrane antibody protein of the present invention. The upper graph is a graph of the expression profile of human prostate-specific membrane antibody protein 9, and the lower graph is the graph of the expression profile of human prostate-specific membrane antibody protein 9.

 Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of human prostate-specific membrane antibody protein 9 isolated. 9KDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention

 The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally in accordance with the general conditions such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Harbor Harbor Labora tory Pres s, 1989), Or as recommended by the manufacturer.

 Example 1: Cloning of human prostate-specific membrane antibody protein 9

Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) mRM was isolated from total RM using Quik mRNA I solat ion Kit (product of Qiegene). 2ug poly (A) mRM forms cDNA by reverse transcription. The Smart 00 cDNA cloning kit (purchased from Clontech) was used to insert the 00 fragment into the multiple cloning site of the pBSK (+) vector (Clontech) to transform DH5 ct. The bacteria formed a CDM library. The sequences of the 5 'and 3' ends of all clones were determined using a Dye terminate cycle reaction ionization kit (Perkin-Elmer) and an ABI 377 automatic sequencer (Perkin-Elmer). CDNA The sequence was compared with the existing public DNA sequence database (Genebank), and the cDNA sequence of one of the clones 0491C07 was found to be new DNA. A series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions. The results show that (the full-length cDNA contained in the H91c07 clone is 3321bp (as shown in Seq IDN0: l), and there is a 246bp open reading frame (0RF) from 1477bp to 1722bp, which encodes a new protein (such as Seq ID NO : Shown in 2). We named this clone pBS-0491c07, and the encoded protein was named human prostate-specific membrane antibody protein 9. Example 2: Cloning the gene encoding human prostate-specific membrane antibody protein 9 by RT-PCR

 CDM was synthesized by reverse transcription reaction using fetal brain cell total MA as a template and oligo-dT as a primer. After purification with Qiagene's kit, PCR was performed using the following primers:

 Primerl: 5, one GAAAAATTAAGATATTATTATAAA -3 '(SEQ ID NO: 3)

 Primer2: 5'- CTCTTGCTGTCTTCCTTTGTGATT -3 '(SEQ ID NO: 4)

 Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;

 Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.

 Conditions for the amplification reaction: 50 μl of KC1, 10 μl / L Tris-

CI, (pH8.5), 1.5mmol / L MgCl ₂ , 200 μmol / L dNTP, lOpmol primer, 1U Taq DNA polymerase (Clontech). The reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72. C 2min. During RT-PCR, β-actin was set as a positive control and template blank was set as a negative control. The amplified product was purified using a QIAGEN kit and ligated to a PCR vector (Invitrogen product) using a TA cloning kit. DM sequence analysis results show that the DM sequence of the PCR product is exactly the same as that of 1 to 3321bp shown in SEQ ID NO: 1. Example 3: Northern blot analysis of human prostate-specific membrane antibody protein 9 gene expression:

Total RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] ₀ This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 time volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1 ), Mix and centrifuge. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. Using 20 μ _§ RNA, electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (H7.0) -5 mM sodium acetate-ImM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane. Preparation ³² P- DM probe labeled with a- ³² P dATP by random priming method. The DNA probe used was the PCR-encoded human prostate-specific membrane antibody protein 9 coding region sequence (1477bp to U22bp) shown in FIG. 1. 32P-labeled probe (about 2 x 10 ⁶ cpm / ral) was transferred with RM The nitrocellulose membrane was hybridized overnight at 42 ° C in a solution containing 50% formamide-25mM H ₂ P0 ₄ (H7.4) -5 χ SSC-5 χ Denhardt's solution and 200 μg / ml salmon Sperm DNA. After hybridization, the filter was placed at 1 x SSC-0.1 ° /. Wash in SDS at 55 ° C for 30 min. Then, Phosphor Imager was used for analysis and quantification. Example 4: In vitro expression, isolation and purification of recombinant human prostate-specific membrane antibody protein 9

 Based on SEQ ID NO: 1 and the coding region sequence shown in Figure 1, a pair of specific amplification primers were designed, the sequence is as follows:

Primer 3: 5'- CATGCTAGCATGGGTGCCACCTCAGCCCTAGAC -3 '(Seq ID No: 5) Primer4: 5,-CCCAAGCTTTTATTGGGTGCTCCAGTCACGAAG -3, (Seq ID No: 6) The 5' ends of these two primers contain Nhel and Hindlll digestion sites, respectively , Followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively. The Nhel and Hindlll restriction sites correspond to the selection on the expression vector plasmid _P ET-28b (+) (Novagen, Cat. No. 69865.3). Sex endonuclease site. PCR was performed using the PBS-0491c07 plasmid containing the full-length target gene as a template. The PCR reaction conditions were as follows: a total volume of 50 μ1 containing 10 pg of pBS-0491c07 plasmid, Primer-3 and Primer-4 were lpmoi, Advantage polymerase Mix (Clontech) 1 μ1, respectively. Cycle parameters: 4 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Nhel and Hindlll were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into E. coli DH5a by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 g / ml), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0491c07) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method. In containing kanamycin (final concentration 30 g / inl) in LB liquid medium, host strain BL21 _(P ET-0491cO7) were cultured to logarithmic growth phase, IPTG was added to a final concentration of lmmol / L at 37 ° C, Continue incubation for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The affinity chromatography column His. Bind Quick Cartridge (product of Novagen) was used to obtain 6 histidine (6His-Tag). The purified human prostate-specific membrane antibody protein 9 was purified. After SDS-PAGE electrophoresis, a single band was obtained at 9 KDa (Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by the Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2. Example 5 Production of anti-human prostate-specific membrane antibody protein 9 antibodies

 The following human prostate-specific membrane antibody protein 9 was synthesized using a peptide synthesizer (product of PE)

NH2-Met-Gly-Ala-Thr-Ser-Ala-Leu-Asp-Pro-Gly-Ala-Met-Val-Pro-Thr- C00H (SEQ ID NO: 7). The polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively. For methods, see: Avrameas, et al. Immunochemi s try, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. A titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used for BLISA to determine the titer of antibodies in rabbit serum. Total IgG was isolated from antibody-positive rabbit sera using protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. The immunoprecipitation method proved that the purified antibody could specifically bind to human prostate-specific membrane antibody protein 9. Example 6: Application of the polynucleotide fragment of the present invention as a hybridization probe

 Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways. For example, the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is determined whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected. Further, the probe can be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissue or pathology. Whether the expression in tissue cells is abnormal.

 The purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method. Acid sequence or a homologous polynucleotide sequence thereof. Filter hybridization methods include dot blotting, Southern imprinting, Northern blotting, and copying methods. They all use the same steps to immobilize the polynucleotide sample to be tested on the filter. These same steps are as follows: The sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer. The pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid. After the hybridization step, the unhybridized probes are removed by a series of membrane washing steps. This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature), so that the hybridization background is reduced and only strong specific signals are retained. The probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention The polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment. In this example, the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.

First, the selection of the probe

The selection of oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention as hybridization probes should follow the following principles and several aspects to be considered: 1. The preferred range of probe size is 18-50 nucleotides;

 2, GC content is 30% -70%, non-specific hybridization increases when it exceeds;

 3. There should be no complementary regions inside the probe;

 4. Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;

5. Whether the preliminary selection probe is finally selected as a probe with practical application value should be further determined by experiments.

 After completing the above analysis, select and synthesize the following two probes:

 Probe 1 (probel), which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):

 5- TGGGTGCCACCTCAGCCCTAGACCCTGGAGCCATGGTTCCT -3 '(SEQ ID NO: 8)

 Probe 1 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt):

 5- TGGGTGCCACCTCAGCCCTACACCCTGGAGCCATGGTTCCT -3 '(SEQ ID NO: 9) For other commonly used reagents and their preparation methods not related to the following specific experimental steps, please refer to the literature: DNA PROBES GH Keller; MM Manak; Stockton Press, 1989 (USA) And more commonly used molecular cloning experiment manual books such as "Molecular Cloning Experiment Guide" (Second Edition 1998) [US] Sambrook et al., Science Press.

 Sample Preparation:

 1.Extract DNA from fresh or frozen tissue,

Procedure: 1) Place fresh or freshly thawed normal liver tissue in a plate immersed in ice and filled with phosphate buffered saline (PBS). Cut the tissue into small pieces with scissors or a scalpel. Keep tissue moist during operation. 2) Centrifuge the tissue at 1,000 g for 10 minutes. 3) Suspend the pellet (about 10 ml / g) with cold homogenization buffer (0.25 moi / L sucrose; 25 ramol / L Tris-HCl, pH 7.5; 25 ol / LnaCl; 25 ramol / L MgCl ₂ ). 4) Homogenize the tissue suspension at 4 ° C at full speed with an electric homogenizer until the tissue is completely broken. 5) Centrifuge at 1000g for 10 minutes. 6) Resuspend the cell pellet (1 to 5 ml per 0.1 g of the original tissue sample), and centrifuge at 1000 g for 10 minutes. 7) Resuspend the pellet with lysis buffer (add 1 ml per 0.1 g of the initial tissue sample), and then follow the following phenol extraction method.

2, DNA phenol extraction method ''

Steps: 1) Wash the cells with 1-10 ml of cold PBS and centrifuge at 1,000 g for 10 minutes. 2) Resuspend the pelleted cells with cold cell lysate (lx 10 ⁸ cells / ml). Use a minimum of 100ul lysis buffer. 3) Add SDS To a final concentration of 1%, if SDS is added directly to the cell pellet before resuspending the cells, the cells may form large clumps that are difficult to break, and reduce the overall yield. This is particularly serious when extracting> 10 ⁷ cells. 4) Add proteinase K to a final concentration of 200ug / ml. 5) Incubate at 50 ° C for 1 hour or shake gently at 37 ° C overnight. 6) Extract with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge in a small centrifuge tube for 10 minutes. The two should be clearly separated, otherwise centrifuge again. 7) Transfer the water phase to a new tube. 8) Extract with an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes. 9) Transfer the aqueous phase containing DM to a new tube. The DNA was then purified and ethanol precipitated.

3.Purification and ethanol precipitation of DM

Steps: 1) Add 1/1 Q volume of 2mol / L sodium acetate and 2 volumes of cold 1Q ethanol to the A solution and mix. Leave at -20 ° C for 1 hour or overnight. 2) Centrifuge for 10 minutes. 3) Carefully aspirate or pour out the ethanol. 4) Wash the pellet with 500ul of 70% cold ethanol and centrifuge for 5 minutes. 5) Carefully aspirate or pour out the ethanol. Wash the pellet with 500ul of cold ethanol and centrifuge for 5 minutes. 6) Carefully aspirate or pour out the ethanol, then invert on the absorbent paper to drain off the residual ethanol. Air dry for 10-15 minutes to allow the surface ethanol to evaporate. Be careful not to allow the pellet to dry completely, otherwise it will be more difficult to re-dissolve. 7) Resuspend the DNA pellet in a small volume of TE or water. Low-speed vortexing or pipetting, with a dropper, while gradually increasing the TE, mixed until fully dissolved DNA, every 1- 5 χ 10 ⁶ cells extracted about plus lul.

 The following steps 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.

8) Add RNase A to the DNA solution to a final concentration of 100ug / ml, and incubate at 37 ° C for 30 minutes. 9) Add SDS and proteinase K, the final concentrations are 0.5% and 100ug / ml, respectively. Incubate at 37 ° C for 30 minutes. 10) Extract the reaction solution with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge for 10 minutes. 11) Carefully remove the aqueous phase and re-extract with an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes. 12) Carefully remove the water phase, add 180 vols of 2 mol / L sodium acetate and 2.5 vols of cold ethanol, mix and let stand at -20 ° C for 1 hour. 13) Wash the precipitate with 70% ethanol and 100% ethanol, air dry, and resuspend the nucleic acid. The process is the same as steps 3-5. 14) Measure A ₂₆ . And A ₂₈ . To check the purity and yield of DM. 15) Store at -20 ° C after dispensing. Preparation of sample film:

 1) Take 4 x 2 pieces of nitrocellulose membrane (NC membrane) of appropriate size, and mark the spotting position and sample number on it with a pencil. Two NC membranes are needed for each probe for later experiments In the step, the film is washed with high-strength conditions and strength conditions, respectively.

 2) Pipette and control 15 microliters each, spot on the sample film, and dry at room temperature.

3) Place on filter paper infiltrated with O. lraol / LNaOH, 1.5mol / LNaCl for 5 minutes (twice), dry and place in filter paper infiltrated with 0.5fflol / L Tris-HC1 (pH7.0), 3mol / LNaCl Allow to dry for 5 minutes (twice). 4) Clamped in clean filter paper, wrapped in aluminum foil, and dried under vacuum at 60-80 ° C for 2 hours.

 Labeling of probes

1) 3 μ IProbe (0.1 OD / 10 μ 1), add 2 μ IKinase buffer, 8-10 uCi y- ³² P-dATP + 2U Kinase, to make up to a final volume of 20 μ 1.

 2) Incubate at 37 ° C for 2 hours.

 3) 1/5 volume bromophenol blue indicator (BPB).

 4) Pass through a Sephadex G-50 column.

5) Collect the first peak before ³² P-Probes are washed out (monitorable).

 6) 5 drops / tube, collect 10-15 tubes.

 7) Monitor the amount of isotope with a liquid scintillator

8) After the collection liquids of the first peak are combined, ³² P-Pn> b _e (the second peak is free γ- ³² P-dATP) to be prepared.

 Pre-hybridization

 Put the sample membrane in a plastic bag, add 3-1 Omg pre-hybridization solution (1 OxDenhardf s; 6xSSC, 0.1 Irag / ml CT DNA (calf thymus DNA).) After sealing the bag, 68 ° C water Rock shake for 2 hours.

 Cross

Cut a corner of the plastic bag, add the prepared probe, seal the bag, and shake it with 42 ^D C water overnight. Wash film:

 Wash the membrane with high intensity.-1) Take out the sample membrane with good hybridization.

 2) 2xSSC, 0.1% SDS, wash at 40 ° C for 15 minutes (twice).

 3) 0.1xSSC, 0.1 SDS, 40. C Wash for 15 minutes (twice).

 4) Wash in 0.1xSSC, 0.1% SDS at 55 ° C for 30 minutes (twice), and dry at room temperature. Low-intensity washing film:

 1) Take out the sample membrane of hybridization.

 2) 2xSSC, 0.1% SDS, 37. C Wash for 15 minutes (twice).

 3) Wash at 37 ° C for 15 minutes (twice) in 0.1xSSC, 0. Γ / oSDS.

 4) 0.1xSSC, 0.1% SDS, 40. Wash for 15 minutes (twice) and dry at room temperature.

X-ray autoradiography:

-70 ° C, X-ray autoradiography (compression time depends on the radioactivity of the hybrid spot). Experimental results:

 The hybridization experiments performed under low-intensity membrane washing conditions showed no significant difference in the radioactive intensity of the above two probes. However, in the hybridization experiments performed under high-intensity membrane washing conditions, the radioactive intensity of probe 1 was significantly stronger than that of hybridization spots. The radioactive intensity of the hybridization spot of the other probe. Therefore, the presence and differential expression of the polynucleotide of the present invention in different tissues can be analyzed qualitatively and quantitatively with the probe 1. Example 7 DNA Microarray

 Gene chip or gene microarray (DM Microarray) is a new technology currently being developed by many national laboratories and large pharmaceutical companies. The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information. The polynucleotide of the present invention can be used as a target DM for gene chip technology for high-throughput research of new gene functions; searching for and screening new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases . The specific method steps have been reported in the literature. For example, refer to the literature DeRi si, JL, Lyer, V. & Brown, P. 0. (1997) Sc ience 278, 680-686. And the literature Hel le, RA, Schema , M., Cha i, A., Sha lom, D., (1997) PNAS 94: 2150-2155.

 (A) spotting

 A total of 4,000 polynucleotide sequences of various full-length cDMs are used as target DNA, including the polynucleotide of the present invention. They were respectively amplified by PCR. After the purified amplified product was purified, the concentration was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between the points is 280 μm. The spotted slides were hydrated, dried, and cross-linked in a purple diplomatic instrument. After elution, the DM was fixed on the glass slide to prepare chips. The specific method steps are widely reported in the literature. The post-spot processing steps of this embodiment are:

 1. Hydration in a humid environment for 4 hours;

 2. 0.2% SDS was washed for 1 minute;

3. Wash twice with ddH ₂ 0 for 1 minute each time;

4. NaBH _{4 is} blocked for 5 minutes;

 5. 95 ° C water for 2 minutes;

 6. 0.2 SDS washes for 1 minute;

7. Rinse twice with ddH ₂ 0;

 8. Dry and store at 25 ° C in the dark for future use.

(Two) probe marking Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) by one-step method, and the mRNA was purified with Ol igotex mRNA Midi Kit (purchased from QiaGen). Cy3dUTP (5-Amino-propargyl-2'-deoxyur idine 5'-triphate coupled to Cy3 f luorescent dye, purchased from Araersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5-Amino-propargy 2) was used. '-deoxyur idine 5'-triphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech Company, labeled the body's specific tissue (or stimulated cell line) mRNA, and purified the probe to prepare a probe. For specific steps and methods, see:

Schena,

M., Shalon, D., Heller, R. (1996) Proc. Nat l. Acad. Sci. USA. Vol. 93: 10614—

 10619. Schena, M., Shalon, Dari., Davis,. W. (1995) Science. 270. (20): 467-480. (3) Hybridization

 Combine the probes from the two tissues with the chips in UniHyb ™ Hybridizat ion

Solut ion (purchased from TeleChera) hybridization solution for 16 hours, washed with a washing solution (1 <SSC, 0.2% SDS) at room temperature, and then scanned with a ScanArray 3000 scanner (purchased from Genera Scanning, USA). The scanned image was analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.

 The above specific tissues (or stimulated cell lines) are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, Arsenic stimulated the L02 cell line and prostate tissue for 1 hour. Draw a graph based on these 13 Cy3 / Cy5 ratios. (figure 1 ) . It can be seen from the figure that the expression profile of human prostate-specific membrane antibody protein 9 and human prostate-specific membrane antibody protein according to the present invention are very similar. Industrial applicability

 The polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.

 As a transmembrane protein, prostate-specific membrane antibody protein has a high expression level in prostate-related tissues, including benign secretory acinar epithelium, prostate epithelial tumors, and prostate cancer cells. It has the functions of both folate hydrolase and neuropeptidase in the body, and is involved in regulating various important metabolic processes.

Studies have found that a variety of prostate-specific membrane antibody proteins have been used in some solid tumors, endothelial cell tumors, Tumor-associated neovascularization and other cells also have higher expression levels. This suggests that it is closely related to the occurrence of malignant diseases such as tumors and related cancers in the body.

 The expression profile of the polypeptide of the present invention is consistent with the expression profile of human prostate-specific membrane antibody protein, and both have similar biological functions. As a transmembrane protein in vivo, it has proteolytic activities of folate hydrolase and neuropeptidase, and its abnormal expression involves tumorigenesis of prostate tissues, prostate-related tissues, and certain solid tumors and endothelial cell tumors. Occurrence, is also important for the metabolism of folic acid and the metabolism of neurotransmitters, and cause related diseases. It can be seen that the abnormal expression of the human prostate-specific membrane antibody protein 9 of the present invention will produce various diseases, especially prostate cancer, benign prostate tumors, other tumors, folic acid deficiency disorders, neuropsychiatric diseases, embryonic developmental disorders, Growth and development disorders, inflammations, and immune disorders. These diseases include, but are not limited to:

 Tumors of various tissues: prostate cancer, benign prostate cancer, stomach cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, colon cancer, melanoma, bladder cancer, uterine cancer, Endometrial cancer, colon cancer, thymoma, nasopharyngeal cancer, laryngeal cancer, tracheal tumors, neuropsychiatric diseases: neuroblastoma, astrocytoma, ependymal tumor, glioblastoma, neurofibromatosis, elderly Dementia, Parkinson's disease, chorea, depression, amnesia, Huntington's disease, epilepsy, migraine, multiple sclerosis, neuromuscular disease, neurodermal syndrome, schizophrenia, depression, neurological decline , Neural tube insufficiency, Brain developmental malformation, Neuron migration disorder

 Embryonic disorders: congenital abortion, cleft palate, limb loss, limb differentiation disorder, atrial septal defect, neural tube defect, congenital hydrocephalus, congenital glaucoma or cataract, congenital deafness, growth and development disorders: mental development Retardation, brain development disorders, skin, fat, and muscular dysplasia, bone and joint dysplasia, various metabolic deficiencies, stunting, dwarfism, Cushing's syndrome, sexual retardation

 Inflammation: chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations

 Immune diseases: Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B-lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome

 The abnormal expression of the human prostate-specific membrane antibody protein 9 of the present invention will also produce certain hereditary, hematological diseases and the like.

The polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially prostate cancer, benign prostate tumors, other tumors, and folic acid. Xie deficiency, neuropsychiatric diseases, embryonic developmental disorders, growth disorders, inflammation, immune diseases, certain hematological diseases, etc. The invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human prostate-specific membrane antibody protein 9. Agonists enhance human prostate-specific membrane antibody protein 9 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers. For example, mammalian cells or a membrane preparation expressing human prostate-specific membrane antibody protein 9 can be cultured with a labeled human prostate-specific membrane antibody protein 9 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.

 Antagonists of human prostate-specific membrane antibody protein 9 include screened antibodies, compounds, receptor deletions, and the like. Antagonists of human prostate-specific membrane antibody protein 9 can bind to human prostate-specific membrane antibody protein 9 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide to render the polypeptide incapable. Play biological functions.

 When screening compounds as antagonists, human prostate-specific membrane antibody protein 9 can be added to bioanalytical assays, and the compounds can be identified by measuring the effect of the compound on the interaction between human prostate-specific membrane antibody protein 9 and its receptors Whether it is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds. Polypeptide molecules capable of binding to human prostate-specific membrane antibody protein 9 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, human prostate-specific membrane antibody protein 9 molecules should generally be labeled.

 The present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies against human prostate-specific membrane antibody protein 9 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.

Polyclonal antibodies can be produced by injecting human prostate-specific membrane antibody protein 9 directly into immunized animals (such as rabbits, mice, rats, etc.). A variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's Adjuvant, etc. Techniques for preparing monoclonal antibodies to human prostate-specific membrane antibody protein 9 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cells Hybridoma technology, EBV-hybridoma technology, etc. Chimeric antibodies that bind human constant regions and non-human-derived variable regions can be produced using existing techniques (Morri son et al, PMS, 1985, 81: 6851). The existing technology for producing single chain antibodies (US Pat No. 4946778) It can also be used to produce single-chain antibodies against human prostate-specific membrane antibody protein 9.

 Antibodies against human prostate-specific membrane antibody protein 9 can be used in immunohistochemical techniques to detect human prostate-specific membrane antibody protein 9 in biopsy specimens.

 Monoclonal antibodies that bind to human prostate-specific membrane antibody protein 9 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.

 Antibodies can also be used to design immunotoxins that target a particular part of the body. For example, human prostate-specific membrane antibody protein 9 High affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.). A common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill human prostate-specific membrane antibody protein 9 positive Cell.

 The antibodies of the present invention can be used to treat or prevent diseases associated with human prostate-specific membrane antibody protein 9. Administration of appropriate doses of antibodies can stimulate or block the production or activity of human pre-gland specific membrane antibody protein 9.

 The invention also relates to a diagnostic test method for quantitative and localized detection of human prostate-specific membrane antibody protein 9 levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of human prostate-specific membrane antibody protein 9 detected in the test can be used to explain the importance of human prostate-specific membrane antibody protein 9 in various diseases and to play a role in the diagnosis of human prostate-specific membrane antibody protein 9 disease.

 The polypeptide of the present invention can also be used for peptide mapping analysis. For example, the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.

Polynucleotides encoding human prostate-specific membrane antibody protein 9 can also be used for a variety of therapeutic purposes. Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by the non-expression or abnormal / inactive expression of human prostate-specific membrane antibody protein 9. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human prostate-specific membrane antibody protein 9 to inhibit endogenous human prostate-specific membrane antibody protein 9 activity. For example, a mutated human prostate-specific membrane antibody protein 9 may be a shortened human prostate-specific membrane antibody protein 9 lacking a signaling functional domain. Although it can bind to downstream substrates, it lacks signaling activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human prostate-specific membrane antibody protein 9. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, and the like can be used to transfer a polynucleotide encoding human prostate-specific membrane antibody protein 9 into a cell. Build Methods for recombinant viral vectors carrying a polynucleotide encoding human prostate-specific membrane antibody protein 9 can be found in the literature (Sambrook, eta l.). In addition, a recombinant polynucleotide encoding human prostate-specific membrane antibody protein 9 can be packaged into liposomes and transferred into cells.

 Methods for introducing a polynucleotide into a tissue or cell include: injecting the polynucleotide directly into a tissue in vivo; or introducing the polynucleotide into a cell through a vector (such as a virus, phage, or plasmid) in vitro, and then Cells are transplanted into the body and so on.

 Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit human prostate-specific membrane antibody protein 9 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RM molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation. Antisense RM and A and ribozymes can be obtained by any existing RNA or DNA synthesis technology. For example, the technology of solid-phase phosphoramidite synthesis of oligonucleotides has been widely used. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RM. This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter. In order to increase the characterization of the nucleic acid molecule, it can be modified in various ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides should use phosphorothioate or peptide bonds instead of phosphodiester bonds.

 The polynucleotide encoding human prostate-specific membrane antibody protein 9 can be used for the diagnosis of diseases related to human prostate-specific membrane antibody protein 9. The polynucleotide encoding human prostate-specific membrane antibody protein 9 can be used to detect the expression of human prostate-specific membrane antibody protein 9 or the abnormal expression of human prostate-specific membrane antibody protein 9 in a disease state. For example, the DM sequence encoding human prostate-specific membrane antibody protein 9 can be used to hybridize biopsy specimens to determine the expression of human prostate-specific membrane antibody protein 9. Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These technical methods are all mature technologies that are publicly available, and related kits are commercially available. A part or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues. Human prostate-specific membrane antibody protein 9 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human prostate-specific membrane antibody protein 9 transcription products.

Detection of mutations in the human prostate-specific membrane antibody protein 9 gene can also be used to diagnose human prostate-specific membrane antibody protein 9-related diseases. Human prostate-specific membrane antibody protein 9 mutant forms include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human prostate-specific membrane antibody protein 9 DM sequences. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect the expression of proteins. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated. The sequences of the invention are also valuable for chromosome identification. The sequence specifically targets a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for labeling chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DM sequences on a chromosome.

 In short, the PCR primers (preferably 15-35bp) can be used to locate the sequence on the chromosomes according to the cDNA). These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.

 PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes. Using the oligonucleotide primers of the present invention, in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization. Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and pre-selection of hybridization to construct chromosome-specific cDNA libraries.

 Fluorescent in situ hybridization (FISH) of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step. For a review of this technique, see Verma et al., Human Chromosomes: a Manua l of Basic Techniques, Pergamon Pres s, New York (1988).

 Once the sequence is located at the exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendelian Inherity in Man (available online with Johns Hopkins University Wetch Medi Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.

 Next, the difference in cDM or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDM sequence-based PCR. Based on the resolution capabilities of current physical mapping and gene mapping technology, the CDM that is accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).

The polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier. These carriers can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof. The composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases. The present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention. Along with these containers, there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders authorize them to be administered to humans by government agencies that manufacture, use, or sell them. In addition, the polypeptides of the invention can be used in combination with other therapeutic compounds.

 The pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration. Human prostate-specific membrane antibody protein 9 is administered in an amount effective to treat and / or prevent a specific indication. The amount and dosage range of human prostate-specific membrane antibody protein 9 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

Claims

Request for Profit

1. An isolated polypeptide-human prostate-specific membrane antibody protein 9, characterized in that it comprises: a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, or an active fragment, analog, or derivative thereof.

2. The polypeptide according to claim 1, characterized in that the amino acid sequence of the polypeptide, analog or derivative has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 2.

 3. The polypeptide according to claim 2, further comprising a polypeptide having an amino acid sequence represented by SEQ ID NO: 2.

 4. An isolated polynucleotide, characterized in that said polynucleotide comprises one selected from the group consisting of: (a) encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 2 or a fragment thereof, an analog thereof; Polynucleotides of derivatives;

 (b) a polynucleotide complementary to polynucleotide (a); or

 (c) a polynucleotide that is at least 70% identical to) or (b).

 5. The polynucleotide according to claim 4, wherein the polynucleotide comprises a polynucleotide encoding an amino acid sequence shown in SEQ ID NO: 2.

 6. The polynucleotide according to claim 4, characterized in that the sequence of the polynucleotide comprises the sequence of positions 1477-1722 in SEQ ID NO: 1 or the sequence of positions 1-3321 in SEQ ID NO: 1. sequence.

 7. A recombination vector containing an exogenous polynucleotide, characterized in that it is a recombination constructed by the polynucleotide according to any one of claims 4-6 and a plasmid, virus or a carrier expression vector Carrier.

 8. A genetically engineered host cell containing an exogenous polynucleotide, characterized in that it is selected from one of the following host cells:

 (a) a host cell transformed or transduced with the recombinant vector of claim 7; or

 (b) a host cell transformed or transduced with a polynucleotide according to any one of claims 4-6.

 9. A method for preparing a polypeptide having human prostate-specific membrane antibody protein 9 activity, characterized in that the method includes:

 (a) culturing the engineered host cell according to claim 8 under conditions that express human prostate-specific membrane antibody protein 9;

 (b) A polypeptide having human prostate-specific membrane antibody protein 9 activity is isolated from the culture.

10. An antibody capable of binding to a polypeptide, characterized in that said antibody is an antibody capable of specifically binding to human prostate-specific membrane antibody protein 9.

11. A class of compounds that mimic or regulate the activity or expression of a polypeptide, characterized in that they are compounds that mimic, promote, antagonize or inhibit the activity of human prostate-specific membrane antibody protein 9.

 12. The compound according to claim 11, characterized in that it is an antisense sequence of a polynucleotide sequence or a fragment thereof as shown in SEQ ID NO: 1.

13. An application of the compound according to claim 11, characterized in that the compound is used for regulating the activity of human prostate-specific membrane antibody protein 9 in vivo and in vitro.

 14. A method for detecting a disease or susceptibility to a disease associated with a polypeptide according to any one of claims 1-3, characterized in that it comprises detecting the expression level of the polypeptide, or detecting the activity of the polypeptide Or detecting a nucleotide variation in a polynucleotide that causes abnormal expression or activity of the polypeptide.

15. Use of the polypeptide according to any one of claims 1-3, characterized in that it is used for screening mimetics, agonists, antagonists or inhibitors of human prostate-specific membrane antibody protein 9; or Identification of peptide fingerprints.

 16. The use of a nucleic acid molecule according to any one of claims 4-6, characterized in that it is used as a primer for a nucleic acid amplification reaction, or as a probe for a hybridization reaction, or for manufacturing a gene chip Or microarray. ,

17. Use of a polypeptide, polynucleotide or compound according to any one of claims 1 to 6 and 11, characterized in that said polypeptide, polynucleotide or mimetic, agonist, antagonist The agent or inhibitor is composed of a safe and effective dose in combination with a pharmaceutically acceptable carrier as a pharmaceutical composition for diagnosing or treating a disease associated with abnormality of membrane protein 9 of human prostate.

18. Use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that the polypeptide, polynucleotide or compound is used for preparing a treatment such as a malignant tumor, Hematological diseases, HIV infection and immune diseases and drugs of various inflammations.