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WO2001068872A1 - Nouveau polypeptide, sous-unite c humaine de la pompe a proton vacuolaire atpase 22, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, sous-unite c humaine de la pompe a proton vacuolaire atpase 22, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001068872A1
WO2001068872A1 PCT/CN2001/000163 CN0100163W WO0168872A1 WO 2001068872 A1 WO2001068872 A1 WO 2001068872A1 CN 0100163 W CN0100163 W CN 0100163W WO 0168872 A1 WO0168872 A1 WO 0168872A1
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Prior art keywords
polypeptide
polynucleotide
triphosphatase
subunit
acylglycoside
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Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc
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Shanghai Biowindow Gene Development Inc
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Priority to AU39110/01A priority Critical patent/AU3911001A/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)

Definitions

  • 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.
  • 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.
  • human hydrogen ion-adenosine triphosphatase 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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:
  • 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
  • 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.
  • 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
  • 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.
  • 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.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof Minute.
  • 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 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.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • 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 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 refers to base pairing by allowing base salt concentration and temperature Polynucleotides bind naturally.
  • sequence 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:
  • 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.
  • isolated refers to the removal of matter from its original environment (for example, its natural environment if it is naturally occurring).
  • 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.
  • 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).
  • 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 .
  • 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.
  • fragment refers 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 ( ⁇ ) 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).
  • a polypeptide sequence such as a leader sequence or a secreted sequence or a sequence or a proteinogen used to purify
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • “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%.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • 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.
  • 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.
  • 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.
  • 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.
  • Q i agene There are many mature techniques for mRNA extraction, and kits are also commercially available (Q i agene).
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). Wait.
  • a method using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-rapid amplification of cDNA ends
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis.
  • promoters are: the lac or trp promoter of E.
  • 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.
  • 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.
  • 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.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • 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 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.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S2 or Sf9 animal cells
  • 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.
  • 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 .
  • transformation can also be performed by electroporation.
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human vacuolar proton-acylglycoside triphosphatase C subunit 22 (Sc ience, 1984; 224: 1431). Generally there are the following steps:
  • 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.
  • a suitable method such as temperature conversion or chemical induction
  • 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.
  • 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
  • polypeptides of the present invention 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.
  • 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.
  • 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.
  • 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.).
  • 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.
  • 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.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • 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.
  • 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.
  • a polynucleotide encoding human vacuolar proton-acylglycoside triphosphatase C subunit 22 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.).
  • 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.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • 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 3 ⁇ 4 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
  • 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.
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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 of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • 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.
  • 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.
  • suitable pharmaceutical carrier 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.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • 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.
  • 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
  • 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
  • ABI 377 automatic sequencer Perkin-Elmer
  • 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.
  • PCR amplification was performed with the following primers:
  • 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 ⁇ 1 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 ⁇ -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
  • RNA extraction in one step [Anal. Biochem 1987, 162, 156-159] rempliThis 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.
  • RNA probe 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 ⁇ 1, and Primer-3 and Primer-4 primers were Opmo 1 and Advantage polymerase Mix (Clontech) 1 ⁇ 1, 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 ⁇ 8 / ⁇ 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.
  • kanamycin final concentration of 30 ⁇ ⁇ / ⁇ 1 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).
  • 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:
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex.
  • hemocyanin and bovine serum albumin 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.
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • the preferred range of probe size is 18-50 nucleotides
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 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):
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • 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.
  • 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.
  • prehybridization solution 10xDenhardt-s; 6xSSC, 0.1 mg / ml CT DM (calf thymus DNA)
  • Gene microarray or gene 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.
  • 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 ⁇ . 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:
  • 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.
  • Cy3dUTP 5-Amino-propargyl-2--deoxyuridine 5--tr iphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech
  • the probes from the above two tissues were hybridized with the chip in a UniHyb TM 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 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.

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Abstract

L'invention concerne un nouveau polypeptide, une sous-unité C humaine de la pompe à proton vacuolaire ATPase 22, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des troubles du métabolisme de l'eau et des électrolytes, des maladies osseuses, des ulcères gastriques, de toutes sortes de tumeurs malignes, des troubles du développement de l'embryon, des inflammations, de l'hémopathie et de l'infection par VIH. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la sous-unité C humaine de la pompe à proton vacuolaire ATPase 22.
PCT/CN2001/000163 2000-03-15 2001-02-26 Nouveau polypeptide, sous-unite c humaine de la pompe a proton vacuolaire atpase 22, et polynucleotide codant pour ce polypeptide Ceased WO2001068872A1 (fr)

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AU39110/01A AU3911001A (en) 2000-03-15 2001-02-26 Novel polypeptide---a human vacuolar H+ acyladenosine triphosphatase C subunit 22 and polynucleotide encoding it

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CN 00114907 CN1313398A (zh) 2000-03-15 2000-03-15 一种新的多肽——人液泡质子－酰苷三磷酸酶c亚基22和编码这种多肽的多核苷酸
CN00114907.5 2000-03-15

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999022006A1 (fr) * 1997-10-28 1999-05-06 Shanghai Second Medical University Cblafc02: sous-unite de h(+)-atpase vacuolaire

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999022006A1 (fr) * 1997-10-28 1999-05-06 Shanghai Second Medical University Cblafc02: sous-unite de h(+)-atpase vacuolaire

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 1 November 2000 (2000-11-01), YUN C.H. ET AL., Database accession no. NP004776 *

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