WO2001068872A1 - Novel polypeptide---a human vacuolar h+ acyladenosine triphosphatase c subunit 22 and polynucleotide encoding it - Google Patents

Abstract

The invention concerns a human vacuolar H+ acyladenosine triphosphatase C subunit 22 and polynucleotide encoding it. The invention also concerns the process of producing the polypeptide by recombinant DNA technique. The methods for treating many diseases e.g. disorders of aquatic electrolytic metabolism, osseous diseases, gastric ulcer, a variety of tumors, disorders of embryo development, inflammation, hemopathy, infection of HIV etc. utilizing the polypeptide are disclosed. The invention discloses the antagonist agains the polypeptide and therapeutic effects thereof. The invention also discloses the uses of the polynucleotide, which encodes a human vacuolar H+ acyladenosine triphosphatase C subunit 22.

Description

 A new polypeptide-human vacuole it-acyl-glycoside triphosphatase C Yam 22 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, human vacuolar proton-acyl glycoside triphosphatase C subunit 22, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. technical background

 It is known that human hydrogen ion-adenosine triphosphatase can "pump" ions / protons into the organelles in cells or make ions / protons cross the plasma membrane of certain cells (such as osteoclasts, renal interstitial cells). . The catalytic site of human hydrogen ion-adenosine triphosphatase is a hexamer composed of three A subunits and three B subunits, which has the function of binding and hydrolyzing ATP; and the other three subunits C, D, And E have a regulating effect. The main function of human hydrogen ion-adenosine triphosphatase is to regulate the pH of local areas in the cell, and it plays an important role in receptor-mediated cytotoxicity, intracellular membrane flow, protein degradation, and coupling transport. Therefore, human hydrogen ion-adenosine triphosphatase has the effects of renal acidification, bone resorption, and stabilization of intracellular pH. It can be seen that the human V-ATPase C subunit, as one of the three regulatory subunits, has the function of regulating human hydrogen ion-adenosine triphosphatase activity.

 Recombinant human V-ATPase C subunit protein or polypeptide has many uses. These uses include, but are not limited to, direct use as a drug to treat diseases caused by hypofunction or loss of V-ATPase C subunits, and for screening antibodies, polypeptides or other ligands that promote or counteract V-ATPase C subunit function. For example, antibodies can be used to activate or inhibit the function of the V-ATPase C subunit. Screening peptide libraries with expressed recombinant V-ATPase C subunit proteins can be used to find therapeutically valuable polypeptide molecules that can inhibit or stimulate the function of V-ATPase C subunits.

 Gene chip analysis revealed that in the thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, arsenic-stimulated L02 In cell lines and prostate tissues, the expression profile of the polypeptide of the present invention is very similar to the expression profile of human vacuolar proton-acyl glycoside triphosphatase C subunit 42, so their functions may also be similar. The present invention is named as human vacuolar proton-acylglycoside triphosphatase C subunit 22.

As mentioned above, the human vacuolar proton-acyl glycoside triphosphatase C subunit 22 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. More human vacuolar proton-acyl glycoside triphosphatase C subunit 22 proteins need to be identified, especially the amino acid sequence of this protein. Newcomer vacuolar proton-acyl glycoside Isolation of the gene encoding the triphosphatase C subunit 22 protein 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 drugs for diseases, so it is important to isolate its coding DNA. Object of the invention

 It is an object of the present invention to provide an isolated novel polypeptide, human vacuolar proton-acylglycoside triphosphatase C subunit 22, and fragments, analogs and derivatives thereof.

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

 Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human vacuolar proton-acylglycoside triphosphatase C subunit 22.

 It is another object of the present invention to provide a genetically engineered host cell comprising a polynucleotide encoding a human vacuolar proton-acylglycoside triphosphatase C subunit 22.

 Another object of the present invention is to provide a method for producing human vacuolar proton-acylglycoside triphosphatase C subunit 22.

 Another object of the present invention is to provide antibodies against the human vacuolar proton-acylglycoside triphosphatase C subunit 22 of the polypeptide of the present invention.

 Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention, human vacuolar proton-acylglycoside triphosphatase C subunit 22.

 Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormal human vacuolar proton-acylglycoside triphosphatase C subunit 22. Summary of invention

 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 D. 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 the polynucleotide (a);

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

More preferably, the sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 602-1204 in SEQ ID NO: 1; and (b) a sequence having positions 1-1 in SEQ ID NO: 1 318-bit sequence. 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 the activity of human vacuolar proton-acylglycoside triphosphatase C subunit 22 protein, which comprises utilizing the polypeptide of the present invention. The invention also relates to compounds obtained by this method.

 The present invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of human vacuolar proton-acylglycoside triphosphatase C subunit 22 protein in vitro, which comprises detecting the polypeptide or a polynucleotide sequence encoding the same in a biological sample. Mutations, or the amount or biological activity of a polypeptide of the invention in a biological sample.

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

 The present invention also relates to the preparation of polypeptides and / or polynucleotides of the present invention for the treatment of cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human vacuolar proton-acylglycoside triphosphatase C subunit 22 Use of medicine.

 Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein. 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 vacuolar proton-acylglycoside triphosphatase C subunit 22 and human vacuolar proton-acylglycoside triphosphatase C subunit 42 according to the present invention. The upper graph is a graph of the expression profile of human vacuolar proton-acylglycoside triphosphatase C subunit 22, and the lower graph is the graph of the expression profile of human vacuolar proton-acylglycoside triphosphatase C subunit 42.

 Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human vacuolar proton-acylglycoside triphosphatase C subunit 22. 22KDa is the molecular weight of the protein. The arrow indicates the isolated protein band. Summary of the Invention

The following terms used in this specification and the claims shall have the following meanings unless specifically stated: "Nucleic acid sequence" refers to oligonucleotides, nucleotides or polynucleotides and fragments or parts thereof, and may also refer to the genome or synthetic DNA or R, they can be single-stranded or double-stranded, representing the sense or antisense strand. Similarly, the term "amino acid sequence" refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof Minute. 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 "variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing 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" means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature. "Replacement" refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.

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

 An "agonist" refers to a molecule that, when combined with human vacuolar proton-acyl glycoside triphosphatase C subunit 22, can cause changes in the protein and thereby regulate the activity of the protein. An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to human vacuolar proton-acyl glycoside triphosphatase C subunit 22.

 "Antagonist" or "inhibitor" refers to an organism that can block or regulate human vacuolar proton-acylglycoside triphosphatase C subunit 22 when combined with human vacuolar proton-acylglycoside triphosphatase C subunit 22 Molecularly active or immunologically active molecule. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that binds human vacuolar proton-acylglycoside triphosphatase C subunit 22.

 "Regulation" refers to a change in the function of human vacuolar proton-acylglycoside triphosphatase C subunit 22, including an increase or decrease in protein activity, changes in binding characteristics, and human vacuolar proton-acylglycoside triphosphatase C subunit 22 Of any other biological, functional or immune properties.

 "Substantially pure" means substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated. Those skilled in the art can purify human vacuolar proton-acylglycoside triphosphatase C subunit 22 using standard protein purification techniques. Substantially pure human vacuolar proton-acylglycoside triphosphatase C subunit 22 produces a single main band on a non-reducing polyacrylamide gel. The purity of the human vacuolar proton-acylglycoside triphosphatase C subunit 22 polypeptide can be analyzed by amino acid sequence analysis.

"Complementary" or "complementary" refers to base pairing by allowing base salt concentration and temperature Polynucleotides bind naturally. For example, the sequence "CT-GA" can be combined with the complementary sequence "GACT". The complementarity between two single-stranded molecules may be partial or complete. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.

 "Homology" refers to the degree of complementarity and can be partially homologous or completely homologous. "Partial homology" refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Nor thern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully 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 identical or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as by the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madi son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Clus ter method (Hi gg ins, DG and PM Sharp (1988) Gene 73: 237-244). _{0 The} Clus ter method will check the distance between all pairs by Groups of sequences are arranged in clusters. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula:

 Number of residues matching between sequence ^ and sequence B

Number of residues in sequence ^-number of interval residues in sequence ^-number of interval residues in sequence ^X

The percent identity between nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art such as Jotun He in (He in J., (1990) Methods in enzymology 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 substitution, for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.

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

 "Derivative" refers to HFP or a chemical modification of its nucleic acid. 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? ^ Its specific An epitope that sexually binds human vacuolar proton-acylglycoside triphosphatase C subunit 22.

 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 matter from its original environment (for example, its natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living thing, 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 such a polynucleotide or polypeptide may be part of a composition. Since the carrier or composition is not a component of its natural environment, they are still isolated.

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

 As used herein, "isolated human vacuolar proton-acylglycoside triphosphatase C subunit 22" means that human vacuolar proton-acylglycoside triphosphatase C subunit 22 is substantially free of other proteins, lipids, Sugars or other substances. Those skilled in the art can purify human vacuolar proton-acylglycoside triphosphatase C subunit 22 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human vacuolar proton-acylglycoside triphosphatase C subunit 22 polypeptides can be analyzed by amino acid sequence.

 The present invention provides a new polypeptide, human vacuolar proton-acylglycoside triphosphatase C subunit 22, 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 can be naturally purified products or chemically synthesized products, or can be produced from prokaryotic or eukaryotic hosts (such as bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.

The invention also includes fragments, derivatives and analogs of human vacuolar proton-acylglycoside triphosphatase C subunit 22. 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 vacuolar proton-acylglycoside triphosphatase C subunit 22 of the present invention. A fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution The amino acid may or may not be encoded by the genetic code; or (Π) this One kind in which a group on one or more amino acid residues is substituted by other groups to include a substituent; or (II) a kind in which a mature polypeptide is mixed with another compound (such as a peptide which prolongs the half-life of the polypeptide A compound, such as polyethylene glycol), or (IV) a polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence or a proteinogen used to purify the polypeptide) formed by fusion of an additional amino acid sequence into a mature polypeptide Sequence). As set forth herein, such fragments, and their derivatives and analogs are considered to be within the knowledge of those skilled in the art. .

 The present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2. The polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1. The polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a full-length polynucleotide sequence of 1 318 bases, and its open reading frame 602-1224 encodes 200 amino acids. According to the comparison of gene chip expression profiles, it was found that this peptide has a similar expression profile to human vacuolar proton-acylglycoside triphosphatase C subunit 42. It can be concluded that the human vacuolar proton-acylglycoside triphosphatase C subunit 22 has human vacuoles. Proton-acylglycoside triphosphatase C subunit 42 functions similarly.

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. DNA can be coding or non-coding. 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 comprising the polypeptide and a polynucleotide comprising 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 can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .

The invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences). The present invention particularly relates to the present invention under strict conditions. The polynucleotide is a polynucleotide that can hybridize. 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, 60'C; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Fi co ll, 42 ° C, etc .; or (3) only between two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.

 The invention also relates to nucleic acid fragments that hybridize to the sequences described above. As used herein, a "nucleic acid fragment" contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nucleotides. Nucleotides or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding human vacuolar proton-acylglycoside triphosphatase C subunit 22.

 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 vacuolar proton-acylglycoside triphosphatase C subunit 22 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 DM sequence from the genomic DNA; 2) chemically synthesizing the D-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 separation of cDM 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 (Q i agene). And the construction of cDNA libraries is also a common method (Sambrook, et al., Moleclar Cloning, A Labora tory Manua, Cold Spring Harbor Labora tory. New York, 1989). Commercially available cDNA libraries are also available, such as different cDNA libraries from Cl on tech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.

These genes can be screened from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of human vacuolar proton-acylglycoside triphosphatase C subunit 22 The level of transcripts; (4) Detecting protein products expressed by genes by immunological techniques or by measuring 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 herein is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).

 In the (4) method, the protein product of human vacuolar proton-acylglycoside triphosphatase C subunit 22 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Wait.

 A method using PCR technology to amplify DNA / RNA (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. In particular, when it is difficult to obtain a 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 isolated and purified by conventional methods such as by gel electrophoresis.

 The polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be measured 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. 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 vacuolar proton-acyl glycoside triphosphatase C subunit 22 coding sequence, and produced by recombinant technology A method of a polypeptide according to the invention.

In the present invention, a polynucleotide sequence encoding human vacuolar proton-acyl glycoside triphosphatase C subunit 22 may be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention. The term "vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al. Gene, 1987, 56: 125); pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized in a host, any plasmid and vector can be used to construct a recombinant expression vector. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements. Methods known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human vacuolar proton-acylglycoside triphosphatase C subunit 22 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DM technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, etal. Mol ecular ar Cloning, a Labora tory Manua l, Cold Spring 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 lac or trp promoter of E. coli; the _PL promoter of lambda phage; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, 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 and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs that 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 control elements (such as promoters, enhancers, etc.) and selectable marker genes.

 In the present invention, a polynucleotide encoding human vacuolar proton-acyl glycoside triphosphatase C subunit 22 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a polynucleotide containing the polynucleotide or the recombinant vector. Genetically engineered host cells. The term "host cell" refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf9; 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 D may be in the exponential growth phase were harvested, treated with CaC l ₂ method used in steps well known in the art. The alternative is to use MgC l ₂ . 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. Using conventional recombinant DM technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant human vacuolar proton-acylglycoside triphosphatase C subunit 22 (Sc ience, 1984; 224: 1431). Generally there are the following steps:

 (1) A polynucleotide (or variant) encoding human human vacuolar proton-acyl glycoside triphosphatase C subunit 22 of the present invention, or a suitable host transformed or transduced with a recombinant expression vector containing the polynucleotide cell;

(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 mediums. 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 recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

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

 Human hydrogen ion-adenosine triphosphatase can “pump” ions / protons into the organelles of cells or make ions / protons cross the plasma membrane of certain cells (eg, osteoclasts, renal interstitial cells). The main function of human hydrogen ion-adenosine triphosphatase is to regulate the pH of a local area in the cell, and it plays an important role in receptor-mediated cytotoxicity, intracellular membrane flow, protein degradation, and coupling transport. Therefore, human hydrogen ion-adenosine triphosphatase has the effects of renal acidification, bone resorption, and stabilization of intracellular pH.

 The polypeptide of the present invention and human hydrogen ion-adenosine triphosphatase are homologous proteins and contain characteristic sequences of the human hydrogen ion-adenosine triphosphatase family, and both have similar biological functions. It mainly regulates the pH of local areas in the cell in the body, and plays an important role in receptor-mediated puffing, intracellular membrane flow, protein degradation, and coupling transport, especially for bone resorption and kidney electrolytes. Metabolism plays an important role, and its abnormal expression is usually closely related to the occurrence of some related disorders of material metabolism disorders, disorders of protein metabolism, and tumors and cancers of related tissues, and produce related diseases.

It can be seen that the abnormal expression of human vacuolar proton-acyl glycoside triphosphatase C subunit 22 in the present invention will produce A variety of diseases, especially water and electrolyte metabolism disorders, bone diseases, gastric ulcers, various tumors, embryonic development disorders, inflammation, these diseases include but are not limited to:

 Water and electrolyte metabolism disorders: metabolic acidosis, metabolic alkalosis, water poisoning, potassium poisoning, sodium poisoning

 Bone diseases: rickets, osteomalacia, limb deformities, osteoid osteoma, chondroma, chondroma, osteoblastoma, chondroblastoma, etc., giant cell tumor of bone, osteosarcoma, chondrosarcoma, Ewing's sarcoma , Myeloma

 Tumors of various tissues: gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, thymic tumor

 Fetal developmental disorders: congenital abortion, cleft palate, limb loss, limb differentiation disorder, atrial septal defect, neural tube defect, congenital hydrocephalus, congenital glaucoma or cataract, congenital deafness

 Inflammation: gastric ulcer, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, cervicitis, various infectious inflammations

 The invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human vacuolar proton-acylglycoside triphosphatase C subunit 22. Agonists enhance human vacuolar proton-acyl glycoside triphosphatase C subunit 22 to 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 vacuolar proton-acylglycoside triphosphatase C subunit 22 and labeled human vacuolar proton-acylglycoside triphosphatase C subunit 22 can be cultured in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.

 Antagonists of human vacuolar proton-acylglycoside triphosphatase C subunit 22 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human vacuolar proton-acylglycoside triphosphatase C subunit 22 can bind to human vacuolar proton-acylglycoside triphosphatase C subunit 22 and eliminate its function, or inhibit the production of the polypeptide, or with the polypeptide The active site binding prevents the polypeptide from performing biological functions.

 When screening compounds as antagonists, human vacuolar proton-acylglycoside triphosphatase C subunit 22 can be added to a bioanalytical assay. The effects of interactions between humans to determine whether a compound is an antagonist. Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds. Polypeptide molecules capable of binding to human vacuolar proton-acylglycoside triphosphatase C subunit 22 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, the human vacuolar proton-acyl glycoside triphosphatase C subunit 22 molecule 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 human vacuolar proton-acyl glycoside triphosphatase C subunit 22 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.

 Polyclonal antibodies can be produced by injecting human vacuolar proton-acylglycoside triphosphatase C subunit 22 directly into immunized animals (such as rabbits, mice, rats, etc.). A variety of adjuvants can be used to enhance the immune response, including But it is not limited to Freund's adjuvant. Techniques for preparing monoclonal antibodies to human vacuolar proton-acyl glycoside triphosphatase C subunit 22 include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497), triple tumor technology , Human B-cell hybridoma technology, EBV-hybridoma technology, etc. Chimeric antibodies that bind human constant regions to non-human variable regions can be produced using existing techniques (Morrison et al., PNAS, 1985, 81: 6851). The existing technology for producing single chain antibodies (U.S. Pat No. 4946778) can also be used to produce single chain antibodies against human vacuolar proton-acyl glycoside triphosphatase C subunit 22.

 Antibodies against human vacuolar proton-acylglycoside triphosphatase C subunit 22 can be used in immunohistochemical techniques to detect human vacuolar proton-acylglycoside triphosphatase C subunit 22 in biopsy specimens.

 Monoclonal antibodies that bind to human vacuolar proton-acyl glycoside triphosphatase C subunit 22 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 vacuolar proton-acylglycoside triphosphatase C subunit 22 high affinity monoclonal antibodies can covalently bind to bacterial or phytotoxins (such as diphtheria toxin, ricin, ormosine, etc.). A common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill human vacuolar proton-acylglycoside triphosphatase C subunit 22 positive cells.

 The antibodies of the present invention can be used to treat or prevent diseases related to human vacuolar proton-acylglycoside triphosphatase C subunit 22. Administration of an appropriate dose of antibody can stimulate or block the production or activity of human vacuolar proton-acylglycoside triphosphatase C subunit 22.

 The invention also relates to a diagnostic test method for quantitative and localized detection of human vacuolar proton-acylglycoside triphosphatase C subunit 22 levels. These tests are well known in the art and include FISH and radioimmunoassays. The level of human vacuolar proton-acylglycoside triphosphatase C subunit 22 detected in the test can be used to explain the importance of human vacuolar proton-acylglycoside triphosphatase C subunit 22 in various diseases and to diagnose humans A disease in which vacuolar proton-acylglycoside triphosphatase C subunit 22 functions.

 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, more preferably mass spectrometry analysis.

The polynucleotide encoding human vacuolar proton-acylglycoside triphosphatase C subunit 22 can also be used for a variety of therapeutic purposes of. Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by the absence or abnormal / inactive expression of human vacuolar proton-acylglycoside triphosphatase C subunit 22. Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant human vacuolar proton-acylglycoside triphosphatase C subunit 22 to inhibit endogenous human vacuolar proton-acylglycoside triphosphatase C subunit 22 active. For example, a variant human vacuolar proton-acylglycoside triphosphatase C subunit 22 may be a shortened human vacuolar proton-acylglycoside triphosphatase C subunit 22, although it may be related to the downstream substrate. Binding, but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human vacuolar proton-acylglycoside triphosphatase C subunit 22. 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 vacuolar proton-acylglycoside triphosphatase C subunit 22 to a cell Inside. A method for constructing a recombinant viral vector carrying a polynucleotide encoding a human vacuolar proton-acylglycoside triphosphatase C subunit 22 can be found in the existing literature (Sambrook, et al.). In addition, recombinant polynucleotide encoding human vacuolar proton-acyl glycoside triphosphatase C subunit 22 can be packaged into liposomes and transferred into cells.

 Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide 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 vacuolar proton-acylglycoside triphosphatase C subunit 22 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RNA molecule that can specifically decompose specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation. Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides. Antisense RNA molecules can be obtained by in vitro or in vivo transcription of the D sequence encoding the RNA. This D sequence has been integrated downstream of the carrier's RNA ¾ synthase promoter. 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 phosphorothioate or peptide bond instead of the phosphodiester bond is used for the ribonucleoside linkage.

The polynucleotide encoding human vacuolar proton-acylglycoside triphosphatase C subunit 22 can be used for diagnosis of diseases related to human vacuolar proton-acylglycoside triphosphatase C subunit 22. A polynucleotide encoding human vacuolar proton-acylglycoside triphosphatase C subunit 22 can be used to detect the expression of human vacuolar proton-acylglycoside triphosphatase C subunit 22 or human vacuolar proton-acylglycoside triphosphate Abnormal Expression of Phosphatase C Subunit 22 For example, the D sequence encoding human vacuolar proton-acylglycoside triphosphatase C subunit 22 can be used to hybridize biopsy specimens to determine the expression status of human vacuolar proton-acylglycoside triphosphatase C subunit 22. Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These technical methods are publicly mature technologies, Related kits are commercially available. Part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also known as a "gene chip") for differential expression analysis and gene diagnosis of genes in tissues. . Human vacuolar proton-acylglycoside triphosphatase C subunit 22 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human vacuolar proton-acylglycoside triphosphatase C subunit 22 transcription products .

 Detection of mutations in the human vacuolar proton-acylglycoside triphosphatase C subunit 22 gene can also be used to diagnose human vacuolar proton-acylglycoside triphosphatase C subunit 22-related diseases. Human vacuolar proton-acylglycoside triphosphatase C subunit 22 mutant forms include point mutations, translocations, deletions, recombinations, and others compared to normal wild-type human vacuolar proton-acylglycoside triphosphatase C subunit 22 DNA sequences Any exceptions etc. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. 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. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these D sequences on a chromosome.

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

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

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

Once the sequence is located at the exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in V. Mckusick, Mendel ian Inheritance in Man (available online with Johns Hopkins University Wetch Medica l Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions. Next, the differences in cDNA or genomic sequences 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 individuals, 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 using cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the cD accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).

 The polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier. These carriers can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof. The composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which 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 produce, use, or sell. 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 vacuolar proton-acylglycoside triphosphatase C subunit 22 is administered in an amount effective to treat and / or prevent a specific indication. The amount and dose range of human vacuolar proton-acyl glycoside triphosphatase C subunit 22 to be 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. Examples

 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 in the following examples are not marked with specific conditions, usually according to conventional conditions such as Sarabrook et al., Molecular Cloning: The conditions described in the Laboratory Manual (New York: Collsp Harbor Harbor Laboratory Pres s, 1989), or Follow the conditions recommended by the manufacturer.

 Example 1 Cloning of human vacuolar proton-acylglycoside triphosphatase C subunit 22

Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) mRNA was isolated from total RNA using Quik raRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA. Directional insertion of cDNA fragments into pBSK (+) using a Sma rt cDNA cloning kit (purchased from C 1 on t ech) DH5 cc was transformed into the multiple cloning site of the vector (Clontech), and the bacteria formed a cDNA library. Dye terminate cycle reaction 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 the existing public D sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 0215b09 was new DNA. A series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions. The results show that the full-length cDNA contained in the 0215b09 clone is 1318 bp (as shown in Seq ID N0: l), and there is a 603 bp open reading frame (0RF) from 602 bp to 1204 bp, which encodes a new protein (such as Seq ID NO : Shown in 2). We named this clone pBS-0215b09 and the encoded protein was named human vacuolar proton-acylglycoside triphosphatase C subunit 22. Example 2 Cloning of a gene encoding human vacuolar proton-acyl glycoside triphosphatase C subunit 22 by RT-PCR method. Using fetal brain cell total RNA as a template and ol igo-dT as a primer for reverse transcription reaction to synthesize cDNA. Qiagene After purification of the kit, PCR amplification was performed with the following primers:

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

 Primer2: 5'- GTCTCTAGAAACTCTTTTATTACA -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, terminal reverse sequence of SEQ ID NO: 1.

Amplification reaction conditions: A reaction volume of 50 μ1 contains 50 mmo l / L KC 1, 10 mmo l / L Tris-HCl, pH 8. 5, 1.5 mmol / L MgCl ₂ , 20 (^ mol / L dNTP, l Opmol primer, 1U Taq DM polymerase (product of Clontech). The reaction was performed on a PE9600 DM thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min. Set β-act in as a positive control and template blank as a negative control at the same time during RT-PCR. Amplification products were purified using a QIAGEN kit and ligated to a pCR vector using a TA cloning kit (Invitrogen product) ). The DNA sequence analysis results show that the DNA sequence of the PCR product is identical to the 1-1318bp shown in SEQ ID NO: 1. Example 3 Northern blot analysis of human vacuolar proton-acylglycoside triphosphatase C subunit 22 gene expression

Total RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] „This method involves acid guanidinium thiocyanate phenol-chloroform extraction. 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M Sodium acetate (pH 4.0) was used to homogenize the tissue, 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) were added, and the mixture was centrifuged. The aqueous phase layer was aspirated and isopropyl alcohol ( 0.8 volume) and the mixture was centrifuged to obtain an RNA pellet. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. Using 20 μ ₈ RNA, containing 20raM 3- (N- Morpholine) Propanesulfonic acid (pH 7, 0)-5 mM sodium acetate-1 mM EDTA-2. 2M formaldehyde on a 1.2% agarose gel. It was then transferred to a nitrocellulose membrane. A- ³² P dATP was used to prepare ³² P-labeled DNA probes by random primers. The DNA probe used was the PCR amplified human vacuolar proton-acylglycoside triphosphatase C subunit 22 coding region sequence (602bp to 1204bp) shown in FIG. 1. A 32P-labeled probe (about 2 x 10 ⁶ cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH ₂ P0 ₄ (pH7. 4)-5 X SSC- 5 X Denhardt's solution and 20 (^ g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 X SSC- 0.1% SDS at 55 ° C for 30 minutes. Then Phosphor Imager was used for analysis and quantification. Example 4 Recombinant human vacuolar proton-acyl glycoside triphosphatase C subunit 22 in vitro expression, isolation and purification According to the sequence of the coding region shown in SEQ ID NO: 1 and Figure 1, design A pair of specific amplification primers was generated, and the sequence is as follows:

Pr imer 3: 5'- CCCCATATGATGCAGCAGGAATGGGAGGTTCTA -3 '(Seq ID No: 5) Pr imer4: 5'- CATGGATCCTTACACAGAATATTGAACACTTGC -3, (Seq ID No: 6) These two primers contain Ndel and BamHI digestion respectively Site, followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively, and the Ndel and BamHI digestion sites correspond to the expression vector plasmid pET-28b (+) (Novagen product, Ca. No. 69865. 3 Selective endonuclease sites on). PCR was performed using the PBS-0215b09 plasmid containing the full-length target gene as a template. The PCR reaction conditions were as follows: 10 pg of pBS-0215b09 plasmid was contained in a total volume of 50 μ1, and Primer-3 and Primer-4 primers were Opmo 1 and Advantage polymerase Mix (Clontech) 1 μ1, respectively. 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 ligation product was transformed into the colibacillus DH5 cx by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 μ ₈ / ιη 1), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0215b09) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method. Containing kanamycin (final concentration of 30μ _§ / ηι1) in LB liquid medium, host strain BL21 _(P ET-0215b09) cultured at 37 ° C to logarithmic phase, IPTG was added to a final concentration of 1 Implicit ol / L. Continue incubation for 5 hours. The cells were collected by centrifugation, and the supernatant was collected by centrifugation, and the supernatant was collected by centrifugation. The affinity chromatography column His s. Bind Quick Car tr idge (product of Novagen) was used to bind 6 histidines (6H s_Tag). By chromatography, the purified human protein vacuole proton-acylglycoside triphosphatase C subunit 22 was obtained. After SDS-PAGE electrophoresis, a single band was obtained at 22 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 vacuolar proton-acylglycoside triphosphatase C subunit 22 antibody A peptide synthesizer (product of PE company) was used to synthesize the following human vacuolar proton-acylglycoside triphosphatase C subunit 22-specific polypeptides:

 NH2-Met-Gln-Gln-Glu-Trp-Glu-Val-Leu-I le-Pro-Ser-His-Ser-Ser-Leu-C00H (SEQ ID NO: 7). The polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex. 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. 15A titer plate coated with 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine the antibody titer in rabbit serum. Total IgG was isolated from antibody-positive rabbit sera using protein A-Sepharose. The peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography. The immunoprecipitation method demonstrated that the purified antibody specifically binds to human vacuolar proton-acylglycoside triphosphatase C subunit 22. Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe

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

The purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by using a filter hybridization method. Acid sequence or a homologous polynucleotide sequence thereof. Filter hybridization methods include dot blotting, Southern blotting, Nor thern blotting, and copying methods. They are all used to fix the polynucleotide sample to be tested on the filter and then hybridize using basically the same steps. 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 site of the sample on the filter is saturated with the carrier and the synthetic polymer. The pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid. After the hybridization step, the unhybridized probes are removed by a series of membrane washing steps. This embodiment utilizes 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. Needle-to-sample hybridization has the strongest specificity and is retained.

 First, the selection of the probe

 The selection of oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:

 1. The preferred range of probe size is 18-50 nucleotides;

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

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

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

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

 Sample Preparation:

 1.Extract DNA from fresh or frozen tissue

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

Steps: 1) Wash cells with 1-1 Oml cold PBS and centrifuge at 1000g for 10 minutes. 2) Resuspend the pelleted cells (1 × ^{10 8} cells / ml) with cold cell lysate and apply a minimum of 100ul lysis buffer. 3) Add SDS to a final concentration of 1%. If SDS is added directly to the cell pellet before resuspending the cells, the cells may form large clumps that are difficult to break, and reduce the overall yield. This is particularly serious when extracting> 10 ⁷ cells. 4) Add proteinase K to a final concentration of 200ug / ml. 5) Incubate at 50 ° C for 1 hour or shake gently at 37 ° C overnight. 6) Extract with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge in a small centrifuge tube for 10 minutes. The two should be clearly separated, otherwise centrifuge again. 7) Transfer the water phase to a new tube. 8) Extract with an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes. 9) Transfer the DNA-containing aqueous phase to a new tube. The DNA was then purified and ethanol precipitated.

 3. Purification of DNA and ethanol precipitation

Steps: 1) Add 1/10 volume of 2mol / L sodium acetate and 2 volumes of cold 100% ethanol to the DNA solution and mix. At -20. C Let stand for 1 hour or overnight. 2) Centrifuge for 10 minutes. 3) Carefully aspirate or pour out the ethanol. 4) Wash the pellet with 500ul of 70% cold ethanol and centrifuge for 5 minutes. 5) Carefully aspirate or pour out the ethanol. Wash the pellet with 500ul of cold ethanol and centrifuge for 5 minutes. 6) Carefully aspirate or pour out the ethanol, then invert on the absorbent paper to drain off the residual ethanol. Air dry for 10-15 minutes to allow the surface ethanol to evaporate. Be careful not to allow the pellet to dry completely, otherwise it will be more difficult to re-dissolve. 7) Resuspend the D pellet with 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 100ug / ml, and incubate at 37 ° C for 30 minutes. 9) Add SDS and protease to the final concentration of 0.5% and 100ug / mU at 37 ° C for 30 minutes. 10) Extract the reaction solution with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge for 10 minutes. 11) Carefully remove the aqueous phase and re-extract with an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes. 12) Carefully remove the water phase, add 180 vols of 2mol / L sodium acetate and 2.5 vols of cold ethanol, and mix well at -20 ° C for 1 hour. 13) Wash the pellet with 70% ethanol and 100% ethanol, air dry, and resuspend the nucleic acid. The process is the same as steps 3-6. 14) Measure A ₂₆ . And A ₂₈ . To detect the purity and yield of DNA. 15) Store at -20 ° C after dispensing.

 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. High strength Conditions and strength conditions wash the film.

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

 3) Place on filter paper infiltrated with 0.1 mol / L NaOH, 1.5 mol / L NaCl for 5 minutes (twice), and dry in 0.5 mol / L Tris-HCl (pH 7.0), 3 mol / L NaCl filter paper for 5 minutes (twice) and allowed to dry.

 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) 3μ1 Probe (0.1OD / Ιθμΐ), add 2μ1 Kinase buffer, 8-10 uCi γ- ³² P-dATP + 2U Kinase to make up to a final volume of 20μ1.

 2) Incubate at 37 ° C for 2 hours.

 3) Add 1/5 volume of bromophenol blue indicator (BPB I

 4) Pass Sephadex G-50 column.

5) Start collecting the first peak before ³² P-Probe washes out (can be monitored by Monitor;).

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

 7) Monitor the amount of isotope with a liquid scintillator

8) The ³² P-Probe (the second peak is free γ- ³² P-dATP) after the collection of the first peak is combined.

 Pre-hybridization

 The sample membrane was placed in a plastic bag, and 3-10 mg of prehybridization solution (10xDenhardt-s; 6xSSC, 0.1 mg / ml CT DM (calf thymus DNA)) was added. After sealing the mouth of the bag, shake at 68 ° C for 2 hours.

 Cross

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

 High-intensity washing film:

 1) Take out the hybridized sample membrane.

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

 3) 0.1xSSC, 0.1% SDS, wash at 40 ° C for 15 minutes (twice).

 4) In 0.1xSSC, 0.1% SDS, wash 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, 37. C wash for 15 minutes (2 times). 3) Wash in 0.1xSSC, 0, 1% SDS at 37 ° C for 15 minutes (twice).

 4) 0.1xSSC, 0.1% SDS, 40. Wash 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:

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

 Gene microarray or gene microarray (D Microarray) is a new technology currently being developed by many national laboratories and large pharmaceutical companies. 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 rapid, 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 si, JL, Lyer, V. & Brown, P. 0. (1997) Sc ience 278, 680-686. And the literature Hel le, RA, Schema, M., Cha i, A., Sha lom, D., (1997) PNAS 94: 2150-2155.

 (A) spotting

 A total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were amplified by PCR respectively. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and a Cartesian 7500 spotter (purchased from Cartesian Company, USA) was used to spot the glass medium. The distance between the points is 280 μηι. The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to D to fix the slides to prepare chips. The specific method steps have been reported in the literature. The sample post-processing steps in this embodiment are:

 1. Hydration in a humid environment for 4 hours;

 2. 0.2% SDS was washed for 1 minute;

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

4. NaBH _{4 is} blocked for 5 minutes; 5. 95. C water for 2 minutes;

 6. 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.

 (Two) probe marking

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), and another ¹ J was separated by reverse transcription. The fluorescent reagent 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 tissue, and the fluorescent reagent Cy5dUTP (5- Amino -propargyl-2'-deoxyur idine 5'-tr iphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech Company, labeled the body's specific tissue (or stimulated cell line) mRNA, and purified the probe to prepare a probe. For specific steps and methods, see:

 Schena, M., Shalon, D., Heller, R. (1996) Proc. Natl. Acad. Sci. USA. Vol. 93: 10614-10619. Schena, M., Sha lon, Dar i., Davi s , RW (1995) Science. 270 (20): 467-480.

 (Three) cross

 The probes from the above two tissues were hybridized with the chip 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 by Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.

 The above specific tissues (or stimulated cell lines) are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, non-starved L02 cell line, Arsenic stimulated the L02 cell line and prostate tissue for 1 hour. Based on these 13 Cy3 / Cy5 ratios, draw a bar graph (Figure 1). It can be seen from the figure that the expression profiles of human vacuolar proton-acylglycoside triphosphatase C subunit 22 and human vacuolar proton-acylglycoside triphosphatase C subunit 42 are very similar.

Claims

^ Profit requirements

1. An isolated polypeptide-human vacuolar proton-acyl glycoside triphosphatase C subunit 22, characterized in that it comprises: a polypeptide having the amino acid sequence shown in SEQ ID NO: 2, or an active fragment of the polypeptide, similar Thing or derivative.

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

 3> The polypeptide according to claim 2, further comprising a polypeptide having 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 shown in SEQ 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 (a) or (b).

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

 6. The polynucleotide according to claim 4, characterized in that the sequence of the polynucleotide comprises a sequence of positions 602-1204 in SEQ ID NO: 1 or a sequence of positions 1-1-318 in SEQ ID NO: 1 sequence.

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

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

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

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

 9. A method for preparing a polypeptide having human vacuolar proton-acyl glycoside triphosphatase C subunit 22 activity, characterized in that the method comprises:

 (a) culturing the engineered host cell according to claim 8 under the condition of expressing human vacuolar proton-acylglycoside triphosphatase C subunit 22;

(b) Isolation of polysaccharides with human vacuolar proton-acylglycoside triphosphatase C subunit 22 activity from the culture Peptide.

 10. An antibody capable of binding to a polypeptide, characterized in that the antibody is an antibody capable of specifically binding to human vacuolar proton-acyl glycoside triphosphatase C subunit 22.

 11. A class of compounds that mimic or regulate the activity or expression of a polypeptide, characterized in that they are compounds that mimic, promote, antagonize or inhibit the activity of human vacuolar proton-acylglycoside triphosphatase C subunit 22.

 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.

 1. Use of the compound according to claim 11, characterized in that the compound is used for a method for regulating the activity of human vacuolar proton-acylglycoside triphosphatase C subunit 22 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 a polypeptide according to any one of claims 1-3, characterized in that it is used for screening a human vacuolar proton-acylglycoside triphosphatase C subunit 22 mimic, agonist, antagonist or Inhibitors; or for peptide fingerprinting.

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

 17. Use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that said polypeptide, polynucleotide or mimetic, agonist, antagonist is used Or the 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 human vacuolar proton-acylglycoside triphosphatase C subunit 22 abnormality.

 18. Use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that said polypeptide, polynucleotide or compound is used for preparing for treating malignant tumors, blood, etc. Disease, HIV infection and immune diseases and drugs of various inflammations.