WO2002012457A1 - A new polypeptide-human protein phosphatase 13.31 and the polynucleotide encoding it - Google Patents

Abstract

The present invention discloses a new polypeptide-human protein phosphatase 13.31, the polynucloetide encoding it and a method producing the polypeptide by DNA recombinant technology. The present invention further discloses a method of the polypeptide treating various disorders, eg., embryonic developmental abnormality, autoimmune disease, tumor, and for studing human antisenescence etc. The present invention also discloses an antagonist of the polypeptide and its therapeutic action. The present invention further discloses the use of the polynucleotide encoding the new human protein phosphatase 13.31.

Description

 A new polypeptide-human protein phosphatase 13.31 and a polynucleotide encoding the polypeptide TECHNICAL FIELD

 The present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, a human protein phosphatase 1 3. 31, and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides. Background technique

 In cell signaling, the most important regulatory mechanism is protein phosphorylation and dephosphorylation. Protein phosphorylation and dephosphorylation are catalyzed by protein kinases and protein phosphatases, respectively. Protein phosphatase is considered paramount because this enzyme shuts down the signaling pathways activated by the kinase, preventing unrestricted transmission of signals. Protein phosphatase is involved in a series of important physiological activities such as regulating enzyme activity, assembling large molecular proteins, intracellular protein distribution, regulating proteolysis, signaling pathways, cell cycle cycling, τ cell activation and neurotransmitter receptor activation. play an important role.

 Protein phosphatase 2C (PP2C) is one of four major Mn2 + or Mg2 + -dependent protein serine / threonine phosphatases. PP2C is a monomeric enzyme with a molecular weight of 42Kd and a low substrate specificity. The catalytic activity depends on the divalent cations (mainly manganese and magnesium ions). Three types of PP2C isoenzymes, PP2C-α, and yah, were found in mammals. There are at least four PP2C homologues in yeast: phosphatase PTC1. In addition to serine phosphatase activity, there is also weak tyrosine phosphatase activity; in addition, there are PTC2, PTC3, and YBR125C.

 The crystal structure of human PP2C shows that the catalytic center consists of a β-sandwich structure, which is combined with two manganese ions surrounded by an α-helix. The water molecules bound by manganese ions interact with the phosphate group of the substrate at the center of the double metal ion and act as an electron acceptor in the dephosphorylation reaction.

 All currently known serine / threonine protein phosphatases are found to be expressed in the brain. PP2C interacts with the activated p38 protein kinase and plays an important role in the nervous signaling pathway. One possible PP2C substrate in neurons is CaMKI I, which is dephosphorylated by PP2C at the autophosphorylation site. Another possible substrate is DARPP-32, which is phosphorylated by tyrosine protein kinase 1.

 Gene chip analysis revealed that in the bladder mucosa, PMA + Ecv 304 cell line, LPS + Ecv304 cell line thymus, normal fibroblasts 1024NC, Fi brob last, growth factor stimulation, 1 024NT, scar-forming fc growth factor stimulation, 1013HT , Scar into fc without stimulation with growth factor, 101 3HC, bladder cancer cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma, polypeptide of the present invention The expression profile is very similar to the expression profile of human protein phosphatase, so their functions may also be similar. The invention is named human protein phosphatase 1 3.31.

As described above, the human protein phosphatase 13.31 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 it has been necessary to identify more involved in these Process of human protein phosphatase 13. 31 protein, especially identifying the amino acid sequence of this protein Column '. Isolation of the novel human protein phosphatase 13.31 protein encoding gene also provides a 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 agents for disease 1 and it is therefore important to isolate its coding DNA. Disclosure of invention

 It is an object of the present invention to provide isolated novel polypeptides-human protein phosphatase 13.31 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 human protein phosphatase 13.31.

 Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human protein phosphatase 13.31.

 Another object of the present invention is to provide a method for producing human protein phosphatase 13.31. '

 Another object of the present invention is to provide an antibody against the polypeptide-human protein phosphatase 13.31 of the present invention. Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors against the polypeptide-human protein phosphatase 13.31 of the present invention.

 Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of human protein phosphatase 13.31.

 The present invention relates to an isolated polypeptide, which is of human origin, and includes: 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 the 31 ⁹ -position in SEQ ID NO: 1; and (b) a sequence having 1-869 in SEQ ID NO: 1 Sequence of bits.

 The invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said 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 present invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit human protein phosphatase 13.31 protein activity, which comprises utilizing the polypeptide of the present invention. The invention also relates to compounds obtained by this method.

The invention also relates to an in vitro detection of a disease or disease associated with abnormal expression of human protein phosphatase 13.31 protein. N01 / 01118 A method for susceptibility to disease, comprising detecting a mutation in said polypeptide or a coding polynucleotide sequence thereof in a biological sample, or detecting the amount or biological activity of a polypeptide of the present invention in a biological sample.

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

 The present invention also relates to the use of the polypeptide and / or polynucleotide of the present invention in the preparation of a medicament for the treatment (cancer, developmental disease or immune disease) or other diseases caused by abnormal expression of human protein phosphatase 13.31.

 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 otherwise:

 "Nucleic acid sequence" refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and may also refer to genomic or synthetic MA or RNA, which may be single-stranded or double-stranded, representing the sense strand or Antisense strand. Similarly, the term "amino acid sequence" refers to an oligopeptide, peptide, polypeptide or protein sequence and a fragment or part 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 acid 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 isoleucine with 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 "immunological activity" refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind to specific antibodies in a suitable animal or cell.

 An "agonist" refers to a molecule that, when combined with human protein phosphatase 13.31, causes a change in the protein to 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 protein phosphatase 13.31.

 An "antagonist" or "inhibitor" refers to a molecule that can block or regulate the biological or immunological activity of human protein phosphatase 13.31 when combined with human protein phosphatase 13.31. Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates, or any other molecule that can bind to human protein phosphatase 1 3.31.

"Regulation" refers to a change in the function of human protein phosphatase 13.31, including an increase or decrease in protein activity, a change in binding characteristics, and any other biological, functional, or immune properties of human protein phosphatase 13.31 Change. ¹¹ Substantially pure '' means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify human protein phosphatase 13.31 using standard protein purification techniques. The substantially pure human protein phosphatase 13.31 produces a single main band on a non-reducing polyacrylamide gel. The purity of human protein phosphatase 13.31 polypeptide can be analyzed by amino acid sequence.

 "Complementary" or "complementary" refers to the natural binding of a polynucleotide 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 can 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 the hybridization of a fully complementary sequence to a target nucleic acid. The 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 that the conditions of reduced stringency allow non-specific binding, because the conditions of 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 the same 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., Mad Son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Higg ins, D. G. and P. M. Sharp (1988) Gene 73: 237-244).

The Cluster method arranges groups of sequences into clusters by checking the distance between all pairs. 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 B

 X 100

The number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B can also be determined by the Cluster method or using methods known in the art such as Jotun Hein. J., (1990) Methods in emzumo l ogy 183: 625-645) ₀

 "Similarity" refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences. 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 DNA or RNA sequence. "Antisense strand" refers to a nucleic acid strand that is complementary to the "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 epitope of human protein phosphatase 13.31.

 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 is not isolated when it is present in a living animal, 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 vector, or it may be such a polynucleotide or peptide that is part of a composition. Since the carrier or composition is not part of its natural environment, they are still separate.

 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 existing in the natural state. .

 As used herein, "isolated human protein phosphatase 13. 31" means human protein phosphatase 13. 31 is substantially free of other proteins, lipids, carbohydrates, or other substances naturally associated with it. Those skilled in the art can purify human protein phosphatase using standard protein purification techniques 13.31. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. Human protein phosphatase 13.31 The purity of the polypeptide can be analyzed by amino acid sequence.

 The present invention provides a new polypeptide, human protein phosphatase 13.31, which is basically composed 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 may be naturally purified products or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. The polypeptides of the present invention may also include or not include the initial cysteine residues.

The invention also includes fragments, derivatives and analogs of human protein phosphatase 13.31. 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 protein phosphatase 13.31 of the present invention. A fragment, derivative, or analog of the polypeptide of the present invention may be: (I) a type 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 one or more of the amino acid residues is substituted with another group to include a substituent; or (III) such One, wherein the mature polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) such that the additional amino acid sequence is fused to the mature polypeptide The resulting polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence used to purify the polypeptide or a protein sequence). As explained herein, such fragments, derivatives, and analogs are considered to be within the scope of those skilled in the art. Inside.

 The present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having an SBQ ID NO: 2 amino acid sequence. 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 869 bases, and its open reading frames 319- 684 encode 121 amino acids. According to the comparison of gene chip expression profiles, it was found that this peptide has a similar expression profile to human protein phosphatase, and it can be deduced that the human protein phosphatase 13.31 has a similar function to human protein phosphatase.

 The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. MA can be coded or non-coded. 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 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 that encodes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.

 The 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 invention. Variants of this polynucleotide may 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 invention also relates to a polynucleotide that hybridizes to the sequence described above (with at least two sequences between

50%, preferably 70% identity). 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 50½ (v / v) formamide, 0.1% calf serum / 0.1 ° / ° F i co ll, 42 ° C, etc .; or (3) only in two sequences Hybridization occurs when the identity between at least 95% and more preferably 97%. Furthermore, 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 in the present invention, a "nucleic acid fragment" contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, 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 protein phosphatase 13.31. 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 protein phosphatase 13.31 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 DNA 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 cDNA 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, and kits are also commercially available (Qiagene). Construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989). Commercially available cMA libraries are also available, such as different cDNA libraries from Clontech. When combined with polymerase reaction technology, even very few 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): (1) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) measuring the level of human protein phosphatase 13.31 transcript; Detection of gene-expressed protein products by immunological techniques or determination of biological activity. The above methods can be used singly 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, immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA) can be used to detect the protein product expressed by the human protein phosphatase 13.31 gene.

 A method for amplifying DNA / RNA by PCR (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 cDNA from a library, the RACE method (RACE-rapid amplification of cDNA ends) can be preferably used. 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 DNA / RNA fragments can be separated 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, sequencing needs to be repeated. Row. Sometimes it is necessary to determine the cDNA 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 human protein phosphatase 13.31, and the recombinant technology to produce the polypeptide of the present invention. Methods.

 In the present invention, a polynucleotide sequence encoding human protein phosphatase 13.31 can 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 well known in the art. Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Ros enberg, etal. Gene, 1987, 56: 125) expressed in bacteria; pMSMD expression vectors expressed in mammalian cells ( Lee and Na thans, 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 in the host, any plasmid and vector can be used to construct a recombinant expression vector. An important feature of expression vectors is that they often contain origins of replication, promoters, marker genes 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 protein phosphatase 13.31 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Mo lecul ar Cloning, a Labora tory Manua l, co l d Harbor Labora tory. New York, 1989). The DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the l ac 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, and the early and late SV40 promoters 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 for translation initiation, a transcription terminator, and the like. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers ^ cis-acting factors expressed by DNA, often about 10 to 300 base pairs, act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenoviral enhancers.

 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 regulatory elements (such as promoters, enhancers, etc.) and selectable marker genes.

In the present invention, a polynucleotide encoding human protein phosphatase 13.31 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute 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: E. coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as flies S2 or Sf 9; animal cells such as CH0, COS or Bowes melanoma cells.

Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA 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 DNA can be harvested after the exponential growth phase and treated with CaCl. The steps used are well known in the art. Alternatively, M _g C l 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.

 By conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant human protein phosphatase 13. 31 (Scence, 1984; 224: 1431). Generally there are the following steps:

 (1) using the polynucleotide (or variant) encoding human human protein phosphatase 13.31 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 media. 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 necessary, the physical, chemical, and other properties can be used to separate and purify the recombinant protein by various separation methods. 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 protein phosphatase 13.31 and human protein phosphatase of the present invention. The upper graph is a graph of the human protein phosphatase 13.31, and the lower graph is the graph of the human protein phosphatase. Among them, 1-fetal brain, 2-bladder mucosa, 3-PMA + Ecv304 cell line, 4-LPS + Ecv304 cell line thymus, 5-normal fibroblasts 1024NC, 6- Fibrob las t, growth factor stimulation, 1024NT, 7 -Scar-fc growth factor stimulation, 1013HT, 8-scar-fc growth factor stimulation without growth factor, 1013HC, 9-bladder cancer cell EJ, 10-bladder cancer, 11-bladder cancer, 12-liver cancer, 13-liver cancer Cell lines, 14-fetus, 15-spleen, 16-prostate cancer, 17-jejunum adenocarcinoma.

Figure 2 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of the isolated human protein phosphatase 13.31. OkDa 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 conventional conditions such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Harbor Labora tory Pres s, 1989), Or as recommended by the manufacturer.

 Example 1: Cloning of human protein phosphatase 13.31

Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) mMA was isolated from total RNA using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA. The Smart cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragments into the multiple cloning site of pBSK (+) vector (Clontech) to transform DH5 cx. The bacteria formed a cDNA library. Dye terminate cycle react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Elmer) were used to determine the sequences at the 5 'and 3' ends of all clones. The determined cDNA sequence was compared with an existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 1099c07 was new DNA. A series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions. The results showed that the 1099C07 clone contained a full-length cDNA of 869 bp (as shown in Seq ID NO: 1), and a 365 bp open reading frame (0RF) from 319 bp to 684 bp, encoding a new protein (such as Seq ID NO : Shown in 2). We named this clone _P BS-1099c07, and the encoded protein was named human protein phosphatase 13.31. Example 2: Cloning of a gene encoding human protein phosphatase 13.31 by RT-PCR

 CDNA was synthesized using fetal brain cell total RNA as a template and ol igo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:

 Pr imerl: 5'- GGGGGGACTGTGGAGCACAGCAAC-3 '(SEQ ID NO: 3)

 Pr imer2: 5'- ACGGGACACTGTTAAGTTTATTCT-3 '(SEQ ID NO: 4)

 Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;

 Pr imer2 is the 3'-end reverse sequence in SEQ ID NO: 1.

Amplification conditions: 50 μl of KC1, 10 legs of ol / L Tr is-CI, (pH 8.5.5), 1.5 mmol / L MgCl ₂ , 200 μ mol / L dNTP, l Opmol primer, 1U Taq DNA polymerase (Clontech). The reaction was performed on a PB9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min. During RT-PCR, β-act in 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) using a TA cloning kit. DNA sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as the 1-869bp shown in SEQ ID NO: 1. Example 3: Northern blot analysis of human protein phosphatase 13.31 gene expression:

Total RNA extraction in one step [Ana l. Biochem 1987, 162, 156-159] ₀ This method includes acidic guanidinium thiocyanate Phenol-chloroform extraction. That is, the tissue is homogenized with 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) are added. ) And centrifuge after mixing. 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 g of RNA, electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-lm EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane. With α- ³² Ρ dATP Preparation ^{32 Ρ-} DNA probe labeled by the random primer method. The DNA probe used was the PCR-encoded human protein phosphatase 13.31 coding region sequence (319bp to 684bp) shown in FIG. 32P-labeled probes (approximately 2 x 10 ⁶ cpm / ml) were hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM H ₂ P0 ₄ (pH7.4)-5 x SSC- 5 x Denhardt, s solution and 200 g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 x SSC-0.1% 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 protein phosphatase 13.31

 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: Primer3: 5,-CATGCTAGCATGGACAAGTGTGTGTGCCTATTG- 3, (Seq ID No: 5)

 Primer4: 5'-CATGGATCCTCATGGATGTAGTAGTTTTCGTAA-3 '(Seq ID No: 6)

The 5 'ends of these two primers contain Mel and BamHI restriction sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene, respectively. The Nde I and BamH I restriction sites correspond to the expression vector plasmid pET- Selective endonuclease site on 28b (+) (Novagen, Cat. No. 69865.3). The PCR reaction was performed using pBS-1099c07 plasmid containing the full-length target gene as a template. The PCR reaction conditions were as follows: 10 pg of pBS-1099c07 plasmid, primers Primer-3 and Primer-4 were included in a total volume of 50 μ1, and (1) was 10 mol, Advantage polymerase Mix (Clontech) 1 μ1. Cycle parameters: 94. C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles. Ndel and BamHI were used to double-digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligated product was transformed into E. coli DH5a by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 μ ^ 1), positive clones were selected by colony PCR method and sequenced. A positive clone (PET-1099c07) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) piySs (product of Novagen) using the calcium chloride method. In containing kanamycin (final concentration of 30 μ g / ml) of LB liquid medium, host strain BL21 _(P ET-1099c07) incubated at 37 ° C to logarithmic phase, IPTG was added to a final concentration of lmmol / L , Continue to cultivate 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 target protein human protein phosphatase was 13.31. After SDS-PAGE electrophoresis, a single band was obtained at 10 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 protein phosphatase 13.31 antibodies

 The following peptides specific to human protein phosphatase 13.31 were synthesized using a peptide synthesizer (product of PE Company):

NH2-Met-Asp-Lys-Cys-Val-Cys-Leu-Leu-Thr-Ala-Phe-Ser-Glu-Arg-Met- 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 as an ELISA to determine antibody titers in rabbit serum. Total AgG was isolated from antibody-positive rabbit serum using protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated Sepharos B column and the anti-peptide antibody was separated from the total IgG by affinity chromatography. The immunoprecipitation method proved that the purified antibody could specifically bind to human protein phosphatase 13.31. 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 tissues or 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 using a filter hybridization method. Acid sequence or a homologous polynucleotide sequence thereof. Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They all use the same steps to hybridize the fixed polynucleotide sample to the filter. These same steps are as follows: The sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer so that the non-specific binding sites of the sample on the filter are saturated with the carrier and the synthetic 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) to reduce the hybridization background and retain only strong specific signals. 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 embodiment, 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.The GC content is 30¾-70%, and the 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 unknown genomic sequences and their complements District into For homology comparison, if the homology with the non-target molecule region is greater than 85% or there are more than 15 consecutive bases, the primary selection probe should generally 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 '-TGGACAAGTGTGTGTGCCTATTGACCGCCTTTTCCGAGCGC— 3' (SBQ ID NO: 8)

 Probe 2 (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'-TGGACAAGTGTGTGTGCCTACTGACCGCCTTTTCCGAGCGC-3 '(SEQ ID NO: 9)

 Please refer to the literature for other commonly listed reagents and their preparation methods related to the following specific experimental procedures: DNA PROBES GH Keller; MM Manak; Stockton Press, 1989 (USA) and more commonly used molecular cloning experiment manual books such as "Molecular Cloning" Guide to Experiments (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. Tissue should be kept moist during operation. 2) Centrifuge the tissue at 1,000 g for 10 minutes. 3) Suspend the precipitate (about 10 ml / g) with cold homogenization buffer (0.25 mol / L sucrose; 25 cryptool / L Tris-HCl, pH 7.5; 25 cryptool / LnaCl; 25mmol / L MgCl ₂ ). 4) at 4. C Homogenize the tissue suspension 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 (add 1-5 ml per 0.1 g of the initial tissue sample), and centrifuge at 1,000 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 phenol extraction method below.

 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) with cold cell lysate pelleted cells were resuspended (1 X 10 ⁸ cells / ml) Minimum application lOOul 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 / m. 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 DNA-containing aqueous phase to a new tube. The DNA was then purified and ethanol precipitated.

 3, DNA purification and ethanol precipitation

Steps: 1) Add 10 volume of 2mol / L sodium acetate and 2 volumes of cold 100% ethanol to the DNA solution and mix. In - ² 0 ° C for 1 hour or overnight to. 2) Centrifuge for 10 minutes. 3) Carefully aspirate or pour out the ethanol. 4) Use 70% Wash the pellet with 500ul of 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 evaporate the surface ethanol. 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 X 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 100 ug / ml, and incubate at 37 ° C for 30 minutes. 9) Add SDS and proteinase K, the final concentration is 0.5% and 100ug / ml. 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 10 vol. 2mol / L sodium acetate and 2.5 vol. Cold ethanol, mix and set 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) Determine A ₂₆ . And A _2M to detect DNA purity and yield. 15) Store at -20 after packing. C.

Preparation of sample film:

 1) Take 4x2 pieces of nitrocellulose membranes (NC membranes) of appropriate size, and mark the spotting position and sample number on it with a pencil. Two NC membranes are required for each probe, so that they can be used in the following experimental steps. The film was 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 impregnated with 0.1 mol / L NaOH, 1.5 mol / L NaCl for 5 minutes (twice), dry and place on filter paper impregnated with 0.5 mol / L Tris-HCl (pH 7.0), 3 mol / L NaCl 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 μl Probe (0.10D / 10 μ 1), add 2 μ IKinase buffer, 8-10 uCi γ- ³² P-dATP + 2U Kinase, to make up to a final volume of 20 μ1.

 2) Incubate at 37 ° C for 2 hours.

 3) Add 1/5 volume of Bromophenol Blue Indicator (BPB).

 4) Pass through a Sephadex G-50 column. ,

5) Before the ³² P-Probe is washed out, start collecting the first peak (can be monitored by Monitor).

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

 7) Monitor the amount of isotope with a liquid scintillator

8) were collected after the first peak were combined as required ³² P-Prob _e prepared (the second peak to the free γ- ³² Ρ- (1ΑΤΡ). Prehybridization

The sample film was placed in a plastic bag, was added ³ - 10mg prehybridization solution (lOxDenhardt's; 6xSSC, 0. lmg / ml CT DNA ( calf thymus DNA).), The sealed bag, 68 ° C shaking water bath for 2 hours .

Cross Cut a corner of the plastic bag, add the prepared probe, seal the bag, and shake it at 42 ° C in a water bath overnight. Wash film:

 High-intensity washing film:

 1) Take out the hybridized sample membrane.

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

 3) Wash in 0.1xSSC, 0.1% SDS at 40 ° C 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 hybridized sample membrane.

 2) 2xSSC, 0.1% SDS, wash at 37 ° C for 15 minutes (twice).

 3) Wash in 0.1xSSC, 0.1% SDS at 37 ° C for 15 minutes (twice).

 4) Wash in 0.1xSSC, 0.1% SDS at 40 ° C for 15 minutes (twice), and dry at room temperature. X-ray auto-development:

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

 Experimental results:

 的 The hybridization experiments performed under low-intensity membrane washing conditions showed no significant difference in the radioactive intensity of the above two probe hybrid spots; while the hybridization experiments performed under high-intensity membrane washing conditions, the radioactive intensity of probe 1 was significantly stronger. To the radioactive intensity of the hybridization spot of another probe. Therefore, probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues. Example 7 DNA Microarray

 Gene chip or DNA microarray is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of fast, efficient, and high-throughput analysis of biological information. The polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen 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, see the literature DeRi s i, J. L., Lyer, V. & Brown, P. 0.

 (1997) Sc ience 278, 680-686. And literature Hel le, R. A., 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 cDNAs are used as target DNA, including the polynucleotide of the present invention. Amplify them separately by PCR, and then adjust the concentration to about 500ng / _Ul after purifying the amplified product. A Cartesian 7500 spotter (purchased from Cartesian Company, USA) was used to spot on the glass medium, and the distance between the spots was 280 μm. The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to fix the DNA on the glass slides 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 1 minute;

 6. Wash with 0.2% SDS for 1 minute;

7. Rinse twice with ddH ₂ 0;

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

 (2) Probe labeling

 Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and the mRNA was purified with Ol igotex mRNA Midi Ki t (purchased from QiaGen). J Cy3dUTP (5-Amino-propargyl-2'-deoxyuridine 5'-tr iphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) was used to label the mRNA of human mixed tissues, and it was tested with fluorescent light! J Cy5dUTP ( 5-Amino-propargyl-2'-deoxyur idine 5'-tr iphate coupled to Cy5 f luorescent dye, purchased from Amersham Phamacia Biotech, was used to label the mRNA of specific tissues (or stimulated cell lines) in the body. needle. For specific steps and methods, see:

 Schena, M., Shalon, D., Heller, R. (1996) Proc. Na t l. Acad. Sci. USA. Vol. 93: 10614-

10619. Schena, M., Shalon, Dar i., Davis, R. W. (1995) Science. 270. (20): 467-480.

 (Three) cross

 The probes from the above two tissues and the chip were respectively hybridized in a UniHyb ™ Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, and a washing solution (1 x SSC, 0.2% SDS) was used at room temperature. After washing, scanning was performed with a ScanArray 3000 scanner (purchased from General Scanning, USA), and the scanned images were 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 fetal brain, bladder mucosa, PMA + Ecv304 cell line, LPS + Ecv304 cell line, thymus, normal fibroblasts 1024NC, Fibroblas t, growth factor stimulation, 102, scar Stimulation of fc growth factor, 1013HT, Scar growth without stimulation of fc, 1013HC, bladder cancer cell EJ, bladder cancer, bladder cancer, liver cancer, liver cancer cell line, fetal skin, spleen, prostate cancer, jejunal adenocarcinoma . Draw a chart based on these 18 Cy3 / Cy5 ratios. (figure 1 ) . It can be seen from the figure that the expression profile of human protein phosphatase 13.31 and human protein phosphatase according to the present invention are very similar. Industrial applicability

 The polypeptide of the present invention, as well as its antagonists, agonists and inhibitors, can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection and immune diseases.

 Human protein phosphatase 2C is one of the four major Mn2 + or Mg2 + -dependent protein serine / threonine phosphatases. It is known that serine / threonine phosphatase regulates the Ras signaling pathway. Abnormal expression in vivo can cause dysfunction of Ras signaling pathway, and then lead to the occurrence of related diseases.

The expression profile of the polypeptide of the present invention is consistent with the expression profile of human protein phosphatase 2C, and both have similar biological functions. Polypeptide of the invention in vivo is one of four major M _g 2+ or Mn2 + - dependent protein serine / threonine phosphatase, conduction process Ra s signaling pathway has a regulatory role. Abnormal expression can cause dysfunction of the Ra s signaling pathway. It is known that eight types of growth factor receptors belong to this type of signaling pathway: epidermal growth factor (EGF) receptor, platelet growth factor (PDGF) receptor, macrophages Colony-stimulating factor (M-CSF) receptor, insulin receptor, insulin-like growth factor-1 (IGF-1) receptor, hepatocyte growth factor (HGF) receptor, nerve growth factor (NGF) receptor, and vascular endothelium Growth factor (VEGF) receptor and so on. In the body, these growth factors have important roles in stimulating cell proliferation, regulating protein synthesis, maintaining neuronal function, and immune regulation. Serine / threonine protein kinase is an important enzyme in the Ras protein signaling pathway. Abnormal expression can cause "blocking" of the Ras protein signaling pathway, cause the dysfunction of the above-mentioned growth factors, and lead to embryonic developmental abnormalities, Occurrence of various tumors, immune deficiency, nervous system dysfunction diseases and diseases related to protein metabolism disorders. These diseases include but are not limited to:

 Common Embryonic Malformations

1. Common deformities of face, neck and limbs:

 1. Cleft lip (most common, with alveolar clefts and cleft palate), cleft palate, facial oblique cleft, cervical pouch, cervical fistula, etc.

2. Common deformities of the limbs:

 1) Missing limbs:

 Horizontal absence (congenital short limbs): no arms, no forearms, no hands, no fingers, no legs, no toes, etc .; longitudinal absences: radial / ulnar abscess of upper extremity, tibia / fibula absent of lower extremity, etc .;

 Seal-like hand / foot deformities, etc .;

 2) Limb differentiation disorder: Absence of a certain muscle or muscle group, joint dysplasia, bone deformity, bone fusion, multi-finger (toe) deformity, and (toe) malformation, horse tellurium varus, etc .;

Common malformations of the digestive system:

 Thyroglossal duct cyst, gastrointestinal atresia or stenosis, ileal diverticulum, umbilical fistula, congenital umbilical hernia, congenital nonganglionic megacolon, impervious anus, abnormal bowel transition, bile duct atresia, circular pancreas, etc

3. Common malformations of the respiratory system: laryngotracheal stenosis / occlusion, tracheoesophageal fistula, hyaline membrane disease, unilateral lung failure, ectopic lung lobe, congenital pulmonary cyst, pulmonary insufficiency, etc .;

4. Common malformations of the urinary system: polycystic kidney, ectopic kidney, horse tellurium, double ureter, umbilical urination, bladder eversion, etc. V. Common malformations of the reproductive system: cryptorchidism, congenital inguinal hernia, double uterus, vaginal atresia, hypospadias, true / false hermaphroditism, testicular feminization syndrome, etc.

6. Common malformations of the cardiovascular system: atrial septal defect, ventricular septal defect, abnormal separation of arterial trunk (dislocation of aorta and pulmonary artery, aortic or pulmonary artery stenosis), open ductus arteriosus, etc.

7. Common malformations of the nervous system: neural tube defects (no cerebral malformations, spina bifida, spinal meningocele, hydrocephalous meningocele), hydrocephalus in / outside the brain, etc .;

8. Common malformations of eyes and ears: missing iris, pupil retention, congenital cataract, congenital glaucoma, unique / non-small eye malformations, congenital deafness, auricular deformities, etc. Common tumors of various tissues:

I. Epithelial tissue:

 Papilloma, squamous cell carcinoma [skin, nasopharynx, larynx, cervix], adenoma (carcinoma) [breast, thyroid], mucinous / serous cystadenomas (carcinoma) [ovary], basal cell carcinoma [head and face Skin], (malignant) polytype adenoma [extending gland], papilloma, transitional epithelial cancer [bladder, renal pelvis], etc .;

Mesenchymal organization:

 Fibrous (sarcoma) [limbs], (Malignant) fibrohistiocytoma [limbs], lipo (sarcoma) [subcutaneous tissue, lower limbs, retroperitoneum], leiomyosarcoma (uterus and gastrointestinal), striated muscle (Sarcoma) [head and neck, genitourinary tract, limbs], hemangio (sarcoma), lymphangioma (sarcoma) [skin, subcutaneous tissue, tongue, lips], bone (sarcoma) [cranium, long bone], (malignant Giant cell tumor [femoral / tibia / upper humerus], cartilage (sarcoma) [hand and foot short bone, pelvis / costal / yin / humerus / scapula], synovial (sarcoma) tumor [knee / ankle / wrist / shoulder / Near elbow joint], (malignant) mesothelioma [thoracic / peritoneal], etc .;

Three. Lymphatic hematopoietic tissue:

 Malignant lymphoma [Neck, mediastinum, mesenteric and retroperitoneal lymph nodes], various leukemias [lymphoid hematopoietic tissue], multiple myeloma [push / thoracic / rib / skull and long bone], etc .;

4. Nervous tissue:

 Nerve fiber (sarcoma) [systemic cutaneous nerve / deep nerve and internal organs], (malignant) schwannoma [nervous of head, neck, limbs, etc.], (malignant) glioblastoma [brain], myeloblastoma [ Cerebellum], (malignant) meningiomas [meninges], ganglioblastoma / neuroblastoma [mediastinum and retroperitoneum / adrenal medulla], etc .; 5. Other tumors:

 Moles, malignant melanoma [skin, mucosa], (malignant) hydatidiform mole, chorionic epithelial cancer [uterus],

(Malignant) Sertoli cell, stromal cell tumor, (Malignant) granulosa cell tumor [ovary, testis], seminoma [testis], asexual cell tumor [ovary], embryonal cancer [testis, ovary], (malignant) Teratoma [ovary, testis, mediastinum and palate tail], etc .;

 Cerebral cortical dysfunction related clinical symptoms:

I. Frontal lobe: dementia, personality changes (frontal frontal), strabismus, writing disability (back middle frontal gyrus), motor loss Language (back of frontal gyrus), lack of smell (bottom of frontal lobe), limb paralysis, convulsions (central frontal gyrus), etc .; 2. parietal lobe: paresthesia (central gyrus), aphasia (left corner gyrus), body Disturbances (right parietal lobe), etc. 3. Temporal lobe: Hookback attack (anterior temporal lobe), sensory / amnestic aphasia (left temporal lobe), hearing impairment (rear superior temporal lobe), etc .;

4. Occipital lobe: hemianopia, hallucinations, visual disagreement, etc.

V. Limbic system: emotional symptoms, memory loss, disturbance of consciousness, hallucinations, etc.

 Disorders related to peripheral nervous system dysfunction

 Peripheral nervous system includes: 12 pairs of brain nerves, 31 pairs of spinal nerves, and autonomic nerves (sympathetic and parasympathetic). Its functional disorders can cause related diseases or / and clinical symptoms. These diseases or / and clinical symptoms include, but are not limited to:

I. Neurological disorders:

 Loss of olfactory taste (olfactory nerve), visual impairment and / or visual field defect (optic nerve), ophthalmoplegia, diplopia, changes in pupil size / reflexes (eye movement nerve, pulley nerve, abductor nerve), facial sensory disorders, masticatory muscles Paralysis, neuroparalytic keratitis (triple nerves), facial paralysis (facial nerves), deafness, tinnitus, vertigo, balance disorders, nystagmus (auditory nerves), hoarseness, difficulty swallowing, pharyngeal reflexes disappeared (glossopharyngeal nerve, vagus nerve), Shoulder drooping, neck / shrug fatigue (collateral nerve), lingual muscle paralysis (sublingual nerve), etc .; 2. Spinal nerve dysfunction:

 1. Sensory disorders: inhibitory sensory disorders (lack of sensation, hyposensory sensation), irritating sensory disorders (hypersensitivity, paresthesia, pain), etc .;

 2. Dyskinesias: Central paralysis (monoplegia, hemiplegia, paraplegia), peripheral paralysis, etc. 3. Autonomic (sympathetic and parasympathetic) functional disorders:

1. Cardio-cerebral vascular system:

 Various arrhythmias, such as early atrial, early ventricular, sinus tachycardia, supraventricular tachycardia, ventricular tachycardia, atrial flutter, atrial fibrillation, sinus bradycardia, sinus arrest, sick sinus syndrome, indoor conduction block, etc .;

 CAD, angina pectoris, myocardial infarction, cardiovascular neurosis, acute heart failure, chronic heart failure, HBP, neurogenic orthostatic hypotension, syncope, cerebrovascular accident, hypotension shock, etc .;

2. Respiratory system:

 Pulmonary edema, respiratory muscle paralysis, respiratory failure, bronchial asthma, etc .;

 3. Celiac disease:

 Nausea, vomiting, flatulence, gastrointestinal colic, biliary colic, renal colic, gastrointestinal obstruction, urinary tract obstruction, acute obstructive cholangitis, acute pancreatitis, chronic pancreatitis, etc .;

Urine retention, enuresis, bladder irritation (frequency, urgency, dysuria), constipation, etc .; Reflux esophagitis, chronic gastritis, peptic ulcer, non-ulcer dyspepsia, neurodiarrhea, etc., gastrointestinal neurosis: globus, psychogenic vomiting, nervous gas, anorexia nervosa, irritable bowel Syndrome, etc.

 4. Endocrine system:

 Diabetes, hypoglycemia, hyperlipidemia, hyperlipoproteinemia, obesity, pheochromocytoma, etc .;

 5. Muscle motor system:

 Myasthenia gravis, periodic paralysis, muscle rigidity, muscle spasm, etc .;

 6. Peripheral vascular disease:

 Raynaud's disease, erythematous limb pain, etc .;

 7. Others: dysmenorrhea, glaucoma, visual impairment and ischemic necrosis of multiple organs, such as renal necrosis (renal failure), liver necrosis, intestinal necrosis, etc .;

 Disorders related to protein metabolism disorders

 Disorders of protein metabolism can affect the following major physiological functions of proteins, which can lead to the occurrence of related diseases: 1. Provide energy for the body to maintain tissue growth, renewal and repair:

 Muscle atrophy, weak limbs, weight loss, severe cases can be "caustic"

2. Produce some physiologically active substances, such as hormones, antibodies, amines, etc .:

 1. Protein peptide hormone dysfunction can cause the following diseases:

 1) Insulin and glucagon: diabetes, hypoglycemia, etc .;

 2) Hypothalamus and pituitary hormones: giant disease, dwarfism, acromegaly, cortisolism (Cushing's syndrome), primary aldosteronism, secondary chronic adrenal insufficiency, hyperthyroidism Hypothyroidism (stingle disease, juvenile hypothyroidism, adult hypothyroidism), male / female infertility, menstrual disorders (functional uterine bleeding, amenorrhea, polycystic ovary syndrome, premenstrual tension syndrome, Menopause syndrome), sexual development disorder, diabetes insipidus, inappropriate antidiuretic hormone secretion syndrome, abnormal lactation, etc .;

 3) parathyroid hormone: hyperparathyroidism, hypoparathyroidism, etc .;

 4) Gastrointestinal hormones: peptic ulcer, chronic indigestion, chronic gastritis, etc .;

 2. Disorders of amine metabolism can cause the following diseases:

 Arrhythmia, shock, insanity, epilepsy, chorea, hepatic encephalopathy (noradrenaline, Y-aminobutyric acid, serotonin, glutamine), motion sickness, type I allergic disease (net Measles, hay fever, allergic rhinitis, skin allergies), peptic ulcer (histamine), hypercholesterolemia (taurine), tumors (polyamines), etc .;

 3. Antibody deficiency is prone to various infections:

 Septicemia, suppurative meningitis, pneumonia, bronchitis, otitis media, pyoderma, etc .;

3. The special physiological effects of certain proteins:

Various hemoglobin diseases (anemia, jaundice, tissue-induced organic acidemia due to hypoxia), various coagulation factor deficiency (bleeding), muscle spasms, muscle forcing, muscle paralysis (actin), hyperlipoproteinemia, etc .; Immune disorders

 1. Defects in cellular immune function can cause various intracellular parasitic infections and various viral infections. These diseases include but are not limited to:

 1) Intracellular parasitic infections: typhoid, paratyphoid (typhoid), tuberculosis (tuberculosis), leprosy (leprosy), wave thermal conductivity (brutella), etc .;

 2) Various viral infectious diseases: such as measles virus (measles, measles bronchitis, pneumonia, otitis media, subacute sclerosing panencephalitis), herpes virus (herpes zoster, chicken pox), etc .;

 3) Other, certain fungal (Candida albicans) infections, etc .;

 2. Humoral immune deficiency can cause various extracellular parasites and various viral infections. These diseases include but are not limited to:

1) Various extracellular parasitic infections: lobular pneumonia, otitis media, mastoiditis, sinusitis, meningitis, sepsis (pneumococcus), epidemic meningococcal (meningococcus), trauma, burns Infection (Pseudomonas aeruginosa), etc .;

 2) Various viral infections: polio virus (poliomyelitis), hepatitis virus (A, B, C, D, E, H, G), etc .;

 3) Others, such as parasite (Pneumocystis carinii) infection, etc .;

3. Immunodeficiency patients are prone to malignant tumors, mainly leukemia and lymphatic tumors (malignant lymphomas [neck, mediastinum, mesenteric and retroperitoneal lymph nodes]).

 4. People with immunodeficiency are prone to autoimmune diseases. SLE, rheumatoid arthritis and malignant anemia are more common;

 In addition, there are diabetes and / or hypoglycemia. In summary, the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of various diseases, such as diseases related to dysfunction of Ras protein signaling pathway and the like.

 The present invention also provides screening compounds to identify increasing (agonist) or repressing (antagonist) human protein phosphatases.

1 3. 31 method of medicament. Agonists enhance human protein phosphatase 1 3. 31 Stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers. For example, mammalian cells or membrane preparations expressing human protein phosphatase 13.31 can be cultured together with labeled human protein phosphatase 13.31 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.

 Antagonists of human protein phosphatase 13. 31 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human protein phosphatase 13.31 can bind to human protein phosphatase 13.31 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot function biologically Learn function.

When screening compounds as antagonists, human protein phosphatase 13.31 can be added to bioanalytical assays to determine whether a compound is a compound by measuring the effect of the compound on the interaction between human protein phosphatase 13.31 and its receptor. Antagonist. In the same way as above for screening compounds, the receptor deficiency functioning as an antagonist can be screened. Lost property and similar. Polypeptide molecules capable of binding to human protein phosphatase 13.31 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. During screening, the 13.31 molecule of human protein phosphatase should generally be labeled.

 The present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies against the human protein phosphatase 13.31 epitope. 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 protein phosphatase 13.31 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. . Techniques for preparing monoclonal antibodies to human protein phosphatase 13.31 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, EBV-hybridization Tumor technology, etc. Chimeric antibodies that bind human constant regions and non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). ₀ Existing techniques for producing single-chain antibodies (US Pat No. .4946778) can also be used to produce single chain antibodies against human protein phosphatase 13.31.

 Anti-human protein phosphatase 13.31 antibody can be used in immunohistochemistry to detect human protein phosphatase 13.31 in biopsy specimens.

 Monoclonal antibodies that bind to human protein phosphatase 13.31 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 protein phosphatase 13.31 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 the antibody with a thiol crosslinker 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 protein phosphatase 13.31-positive cells.

 The antibodies of the present invention can be used to treat or prevent diseases related to human protein phosphatase 13.31. Administration of appropriate doses of antibodies can stimulate or block the production or activity of human protein phosphatase 13.31.

 The invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human protein phosphatase 13.31. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of human protein phosphatase 13.31 detected in the test can be used to explain the importance of human protein phosphatase 13.31 in various diseases and to diagnose diseases in which human protein phosphatase 13.31 plays a role.

 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.

The polynucleotide encoding human protein phosphatase 13.31 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 protein phosphatase 13.31. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutant human protein phosphatase 13.31 to inhibit endogenous human protein phosphatase 13.31 activity. For example, a variant human protein phosphatase 13.31 Human protein phosphatase 13.31, which can be shortened and lack the signaling domain, can bind to downstream substrates but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human protein phosphatase 13.31. Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human protein phosphatase 13.31 into a cell. A method for constructing a recombinant viral vector carrying a polynucleotide encoding human protein phosphatase 13.31 can be found in the existing literature (Sambrook, et al.). In addition, a recombinant polynucleotide encoding human protein phosphatase 13.31 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 in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.

 Oligonucleotides (including antisense RNA and DNA) and ribozymes that inhibit human protein phosphatase 13.31 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that specifically decomposes a specific MA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation. Antisense RNA and MA and ribozymes can be obtained by any existing MA or DNA synthesis technology. For example, solid-phase phosphoramidite chemical synthesis technology has been widely used for the synthesis of oligonucleotides. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of MA sequences encoding the RNA. This DNA sequence has been integrated downstream of the MA polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.

 The polynucleotide encoding human protein phosphatase 13.31 can be used for the diagnosis of diseases related to human protein phosphatase 13.31. The polynucleotide encoding human protein phosphatase 13. 31 can be used to detect the expression of human protein phosphatase 13. 31 or the abnormal expression of human protein phosphatase 1 3. 31 in a disease state. For example, a DNA sequence encoding human protein phosphatase 13. 31 can be used to hybridize biopsy specimens to determine the expression of human protein phosphatase 13.31. Hybridization techniques include Sou thern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, 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 (MAcroarray) or an MA chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues. Human protein phosphatase 1 3. 31 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human protein phosphatase 1 3. 31 transcripts.

 Detection of mutations in the human protein phosphatase 13.31 gene can also be used to diagnose human protein phosphatase 13.31-related diseases. Human protein phosphatase 1 3. 31 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to normal wild-type human protein phosphatase 13. 31 DNA 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. Only a few chromosome markers based on actual sequence data (repeating polymorphisms) are now available for labeling chromosomes Location. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.

 In short, PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be mapped on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells containing human genes corresponding to the primers 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, sublocalization can be achieved using a set of fragments from a specific chromosome or a large number of genomic clones. Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection 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 Inheritance in Man (available online with Johns Hopkins University Wetch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that are mapped to chromosomal regions. 'Next, the cDNA or genomic sequence differences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individual, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA 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 contains 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 invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention. Along with these containers, there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body 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 protein phosphatase 13. 31 is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of human protein phosphatase 13.31 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 Rights

I. An isolated polypeptide-human protein phosphatase 13. 31, 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, characterized in that it comprises a polypeptide having the amino acid sequence shown in SEQ ID NO: 2.

 4. An isolated polynucleotide, characterized in that said polynucleotide comprises one selected from the group consisting of:

 (a) a polynucleotide encoding a polypeptide having an amino acid sequence represented by SBQ ID NO: 2 or a fragment, analog, or derivative thereof;

 (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, wherein the sequence of said polynucleotide comprises the sequence of positions 319-684 in SEQ ID NO: 1 or the sequence of positions 1-869 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 the polynucleotide according to any one of claims 4-6.

9. A method for preparing a polypeptide having human protein phosphatase 13.31 activity, characterized in that the method comprises: (a) culturing the project according to claim 8 under the condition of expressing human protein phosphatase 13.31 Host cells

(b) Isolating a polypeptide with human protein phosphatase 13.31 activity 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 protein phosphatase 13.31.

II. 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 protein phosphatase 13.31.

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. The use of the compound according to claim 11, characterized in that the compound is used for regulating the activity of human protein phosphatase 13.31 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. The use of the polypeptide according to any one of claims 1-3, characterized in that it is used to screen a mimetic, agonist, antagonist or inhibitor of human protein phosphatase 13.31; 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 as claimed in any one of claims 1-6 and 11, characterized in that said polypeptide, polynucleotide or mimetic, agonist, antagonist The agent or inhibitor is composed of a safe and effective dose with a pharmaceutically acceptable carrier as a pharmaceutical composition for diagnosing or treating a disease associated with the abnormality of human protein phosphatase 13.31.

 18. The use of a polypeptide, polynucleotide or compound as claimed in any one of claims 1-6 and 11, characterized in that said polypeptide, polynucleotide or compound is used for preparing a treatment such as a malignant tumor, Hematology, HIV infection and immune diseases and drugs of various inflammations.