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WO2002000827A2 - Nouveau polypeptide, proteine de methylation d'adn-cysteine methyl-transferase 10, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine de methylation d'adn-cysteine methyl-transferase 10, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002000827A2
WO2002000827A2 PCT/CN2001/000943 CN0100943W WO0200827A2 WO 2002000827 A2 WO2002000827 A2 WO 2002000827A2 CN 0100943 W CN0100943 W CN 0100943W WO 0200827 A2 WO0200827 A2 WO 0200827A2
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Prior art keywords
polypeptide
protein
polynucleotide
dna
cysteine methyltransferase
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PCT/CN2001/000943
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English (en)
Chinese (zh)
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WO2002000827A3 (fr
Inventor
Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU93611/01A priority Critical patent/AU9361101A/en
Publication of WO2002000827A2 publication Critical patent/WO2002000827A2/fr
Publication of WO2002000827A3 publication Critical patent/WO2002000827A3/fr

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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
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel peptide-methylation-DNA-protein-cysteine methyltransferase 10, and a polynucleotide sequence encoding the polypeptide. The invention also relates to methods and applications for preparing such polynucleotides and polypeptides. Background technique
  • DNA molecules are required to maintain a high degree of accuracy and integrity.
  • organisms have evolved a series of repair systems.
  • the main mutagenic and carcinogenic effect of methylation reagents on DM is the formation of 06-alkylguanine.
  • Repair of DNA containing 06-methylguanine is performed by the enzyme 6-0-methylguanine-DM methyltransferase (methylated-DM-protein-cysteine methyltransferase) (MGMT) .
  • MGMT methylated-DM-protein-cysteine methyltransferase
  • the alkyl group at position 0-6 is transferred to a cysteine residue in the enzyme (Lindahl T., Sedgwick B., Sekiguchi M., Nakabeppu Y., Annu. Rev. Biochem. 57: 133- 157 (1988)) 0
  • Most, but not all, MGMTs also repair 0-4 methylthymine. MGMT exists in different prokaryotic and eukaryotic organisms. The regions around the cysteine residues of the active sites of these enzymes are conserved and can be used as a characteristic sequence template for this family: [LIVMF]-PC- H- R- [LIVMF] (2).
  • point mutations such as the ras, p53 and ERCC-3 genes. These mutations are extremely important for activating (oncogenes) or suppressing (tumor suppressor genes) these genes.
  • Point mutations caused by the action of alkylating agents such as N-nitroso compounds
  • alkylating agents such as N-nitroso compounds
  • the level of this enzyme in cells is considered to be a key factor in determining the sensitivity of cells to the tumor mutagenic effects of alkylating carcinogens (Lindahl et al, Annu. Rev. Biochem. (1988) 57, 133-157; AE Pegg, Cancer Res. (1990) 50, 6119-6129).
  • the new polypeptide of the present invention has a high degree of homology and similarity in structure and function with members of the methylation-DM-protein-cysteine methyltransferase family, and the amino acid sequence also contains the above-mentioned characteristic sequence template It is considered to belong to the methylation-DM-protein-cysteine methyltransferase family.
  • the polypeptide and its agonists, inhibitors and antagonists can be used to diagnose and prevent various cancers caused by alkylated carcinogens (Rafferty JA, Elder RH, Watson AJ, Cawkwe 11 L., Potter PM, Margison GP, Nucleic Acids Res. 20: 1891-1895 (1992)). Name: Methylated-DM-Protein-Cysteine Methyltransferase 10
  • the methylation-DNA-protein-cysteine methyltransferase 10 protein plays an important role in important functions in the body, and it is believed that a large number of proteins are involved in these regulatory processes, so identification in the art has been required. More methylated-DM-protein-cysteine methyltransferase 10 proteins involved in these processes, especially the amino acid sequence of this protein was identified.
  • the isolation of the neomethylated-DM-protein-cysteine methyltransferase 10 protein encoding gene also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for developing diagnostic and / or therapeutic drugs for diseases, so isolating its coding DNA is important. Disclosure of invention
  • An object of the present invention is to provide an isolated novel polypeptide mono-methylated-DNA-protein-cysteine methyltransferase 10 and fragments, analogs and derivatives thereof.
  • -Another object of the present 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 methylated-DM-protein-cysteine methyltransferase 10.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding methylated-DM-protein-cysteine methyltransferase 10.
  • Another object of the present invention is to provide a method for producing methylated-DM-protein-cysteine methyltransferase 10.
  • Another object of the present invention is to provide an antibody against the mono-methylated-DM-protein-cysteine methyltransferase 10 of the polypeptide of the present invention.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors against the mono-methylation-DNA-protein-cysteine methyltransferase 10 of the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities in methylation-DM-protein-cysteine methyltransferase 10.
  • the present invention relates to an isolated polypeptide, which is of human origin, and includes: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • 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:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) having SBQ ID NO: 1 A sequence of positions 366-632; and (b) a sequence of positions 1-645 in SEQ ID NO: 1.
  • 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.
  • 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
  • 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 invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit methylation-MA-protein-cysteine methyltransferase 10 protein activity, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or disease susceptibility related to abnormal expression of methylation-DNA-protein-cysteine methyltransferase 10 protein in vitro, which comprises detecting the polypeptide or its encoding in a biological sample. A mutation in a polynucleotide sequence, or the amount or biological activity of a polypeptide of the invention in a biological sample is detected.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the present invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to polypeptides and / or polynucleotides of the present invention prepared for use in the treatment of cancer, developmental or immune diseases, or other diseases due to abnormal expression of methylation-DNA-protein-cysteine methyltransferase 10 Use of medicines that cause disease.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, 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.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino acid or nucleotide changes or a polynucleotide sequence encoding it. The changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence. Variants can have "conservative" changes, in which the substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • insertion or “addition” is meant 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 naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Biological activity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • 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 methylation-DNA-protein-cysteine methyltransferase 10, causes the protein to alter its plexus and regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind methylated-DNA-protein-cysteine methyltransferase 10.
  • Antagonist refers to a methylation-DM-protein-cysteine that blocks or regulates methylation-MA-protein-cysteine methyltransferase 10 when combined with methylation-MA-protein-cysteine methyltransferase 10
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind methylated-DM-protein-cysteine methyltransferase 10.
  • Regular refers to changes in the function of methylation-DM-protein-cysteine methyltransferase 10, including an increase or decrease in protein activity, changes in binding characteristics, and methylation-DM-protein-hemistein Changes in any other biological, functional, or immune properties of cystine methyltransferase 10.
  • 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 methylated-DNA-protein-half using standard protein purification techniques. Cystine methyltransferase 10. Essentially pure methylation-DM-protein-cysteine methyltransferase 10 produces a single main band on a non-reducing polyacrylamide gel. Methylation The purity of the -DM-protein-cysteine methyltransferase 10 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of a polynucleotide by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence "C-T-G-A” can be combined with the complementary sequence "G-A-C-T”.
  • the complementarity between two single-stranded molecules can be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands. '
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. Suppression of this hybrid.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are 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., Madison Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Cluster method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). The Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. Then cluster each cluster in pairs or Group allocation. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by:
  • the number of residues matching between sequence A and sequence X 100 The number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B can also be determined by the Clus ter method or by methods known in the art For example, Jotun Hein determines the percent identity between nucleic acid sequences (Hein J., (1990) Methods in emzumology 183: 625-645).
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DM or RM sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to the “sense strand”.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? ⁇ It can specifically bind to the epitope of methylation-DNA-protein-cysteine methyltransferase 10.
  • 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 a substance from its original environment (for example, its natural environment if it occurs naturally).
  • a naturally-occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist in the natural system.
  • Such a polynucleotide may be part of a vector, or it may be such a polynucleotide or peptide that is part of a composition. Since the carrier or composition is not part of its natural environment, they are still separate.
  • 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 existing in the natural state. .
  • isolated methylation-DM-protein-cysteine methyltransferase 10 refers to methylation
  • -DNA-protein-cysteine methyltransferase 10 is substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify methylated-DM-protein-cysteine methyltransferase 10 using standard protein purification techniques. Essentially pure peptides can be produced on non-reducing polyacrylamide gels Single main band. The purity of the methylated-DM-protein-cysteine methyltransferase 10 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a novel polypeptide-monomethylated-MA-protein-cysteine methyltransferase 10, 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 (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant techniques.
  • polypeptide of the invention may be glycosylated, or it may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the present invention also includes fragments, derivatives and analogs of methylated-DM-protein-cysteine methyltransferase 10.
  • fragment refers to substantially maintaining the same biological function of the methylated-DNA-protein-cysteine methyltransferase 10 of the present invention or Active peptide.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a type in which one or more amino acid residues are replaced 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 a genetic codon; or ( ⁇ ) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or ( ⁇ ⁇ ) Such a polypeptide sequence in which the mature polypeptide is fused with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol); or (IV) a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide (Such as leader sequences or secretory sequences or sequences used to purify this polypeptide or protease sequences) As set forth herein, such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 645 bases in length and its open reading frame (366-632) encodes 87 amino acids.
  • This polypeptide has a characteristic sequence of methylation-DNA-protein-cysteine methyltransferase, and it can be deduced that the methylation-DNA-protein-cysteine methyltransferase 10 has methylation-DNA Structure and function represented by -protein-cysteine methyltransferase.
  • the polynucleotide of the present invention may be in the form of DM or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DM can be single-stranded or double-stranded.
  • the DM can be a coding chain or a non-coding chain.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • 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) And non-coding sequences.
  • polynucleotide encoding a polypeptide refers to a polynucleotide that encodes the polypeptide and a polynucleotide that includes additional coding and / or non-coding sequences.
  • the present invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention.
  • Variants of this polynucleotide may 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 invention particularly relates to polynucleotides that are hybridizable to the polynucleotides of the invention under stringent conditions.
  • “strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60V; or (2) during hybridization Add denaturants, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficol l, 42 ° C, etc .; or (3) only the identity between the two sequences Crosses occur at least above 95%, more preferably above 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, most preferably at least 100 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding methylated-DNA-protein-cysteine methyltransferase 10.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form and are more preferably purified to homogeneity.
  • polynucleotide sequence encoding the methylated-DM-protein-cysteine methyltransferase 10 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 CDM libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DM 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 DNA sequence to obtain the double-stranded DM of the polypeptide.
  • genomic DM is the least commonly used. Direct chemical synthesis of DM sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating a CDM of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • the construction of cDNA libraries is also a common method (Sambrook, et al, Molecular Cloning, A Laboratory Manua, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cMA libraries are also available, such as different cDNA libraries from Clontech. when When combined with polymerase reaction technology, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DM-DNA or DM-RNA hybridization; (2) the presence or loss of marker gene function; (3) determination of methylation-DM-protein-cysteine methyl The level of transcript of transferase 10; (4) Detecting the protein product of gene expression by immunological techniques or measuring biological activity. The above methods can be used alone 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 2,000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually a DM 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 methylated-DNA-protein-cysteine methyltransferase 10 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunoassay. Adsorption method (ELISA) and so on.
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunoassay.
  • ELISA Adsorption method
  • a method using PCR technology to amplify DNA / RM (Saik i, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-rapid amplification of cDNA ends
  • the primers 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 DM fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits. In order to obtain the full-length cDNA sequence, sequencing must 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 a polynucleotide of the present invention, and to a host cell genetically engineered using the vector of the present invention or directly using a methylation-DNA-protein-cysteine methyltransferase 10 coding sequence. , And a method for producing the polypeptide of the present invention by recombinant technology.
  • a polynucleotide sequence encoding methylated-DNA-protein-cysteine methyltransferase 10 can 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 (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 MA sequences encoding methylated-DM-protein-cysteine methyltransferase 10 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc. (Sambroook, et al. Molecular Cloning, a Laboratory Manua 1, cold Cold Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mMA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, Retroviral LTRs and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells.
  • Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus 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 methylated-DNA-protein-cysteine methyltransferase 10 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute the polynucleotide.
  • a host cell refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DM sequence according to the present invention or a recombinant vector containing the DM sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote, such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DM transfection methods can be used: calcium phosphate co-precipitation method, or conventional machinery Methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant methylated-DM-protein-cysteine methyltransferase 10 (Scence, 1984; 224: 1431) .
  • methylated-DM-protein-cysteine methyltransferase 10 Scence, 1984; 224: 14311
  • the medium used in the culture may be selected from various conventional mediums according to the host cells used. 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, recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.
  • 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
  • Figure 1 is a methylation-DM-protein-cysteine methyltransferase 10 of the present invention at 7-52 with a total of 46 amino acids and methylation-DNA-protein-cysteine methyltransferase characteristic domains Comparison of amino acid sequence homology.
  • the upper sequence is methylated-DNA-protein-cysteine methyltransferase 10
  • the lower sequence is the characteristic domain of methylated-DNA-protein-cysteine methyltransferase.
  • Identical amino acids are represented by a single character amino acid between the two sequences, and similar amino acids are represented by "+".
  • Figure 2 is the polyacrylamide gel electrophoresis of isolated methylation-MA-protein-cysteine methyltransferase 10 (SDS-PAGE ⁇ lOkDa is the molecular weight of the protein.
  • SDS-PAGE ⁇ lOkDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein strip The best way to implement the invention
  • the sequence of the methylation-DNA-protein-cysteine methyltransferase 10 of the present invention and the protein sequence encoded by the methylation-DNA-protein-cysteine methyltransferase 10 were profiled by the prog i le scan program in GCG (Basicloca l Al ignment search tool) [Al tschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], domain analysis was performed in a database such as prosit.
  • the methylation-DNA-protein-cysteine methyltransferase 10 of the present invention is homologous with the domain methylation-DNA-protein-cysteine methyltransferase at 7-52, and the homology results Shown in Figure 1, the homology is 0.28 and the score is 12.91; the threshold is 11.10.
  • Example 3 Cloning of a gene encoding methylation-DNA-protein-cysteine methyltransferase 10 by RT-PCR method Using fetal brain cells total RM as template and ol igo-dT as primer for reverse transcription reaction CDNA synthesis
  • PCR amplification was performed with the following primers:
  • Pr imerl 5 -CATCCTGAGAACTGAAATTGATCGC-3 '(SEQ ID NO: 3)
  • Pr imer2 5'-ATAAAATTTTTGAATTTATGTTCAA-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 conditions 50 mmol / L KC1, 10 mmol / L in a 50 ⁇ l reaction volume Tris s-Cl, (pH 8. 5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol primer, 1 ⁇ Taq polymerase (product of Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • RT-PCR set ⁇ -act in as a positive control and template blank as a negative control.
  • the amplified product was purified using a QIAGEN kit, and ligated to a PCR vector using a TA cloning kit (Invitrogen). DM sequence analysis results showed that the DM sequence of the PCR product was exactly the same as the 1-645bp shown in SEQ ID NO: 1.
  • Example 4 Nor thern blot analysis of methylated-DM-protein-cysteine methyltransferase 10 gene expression:
  • RNA extraction in one step [Ana l. Biochera 1987, 162, 156-159] 0
  • This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) Centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The obtained RM precipitate was washed with 70% ethanol, dried and dissolved in water.
  • a 32P-labeled probe (approximately 2 x 10 6 c P m / 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 200 ⁇ g / ml salmon sperm DM. After hybridization, the filters were placed in 1 x SSC-0. 1% SDS at 55. C Wash for 30min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation, and purification of recombinant methylation-MA-protein-cysteine methyltransferase 10 Based on SBQ ID NO: 1 and the coding region sequence shown in Figure 1, a pair of specific pairs was designed Sexual amplification primer, the sequence is as follows:
  • Pr imer 3 5'-CCCCATATGATGCTCTGTCACCTTCAAAGGATGG-3 '(Seq ID No: 5)
  • Pr imei-4 5 -CCCAAGCTTCTTCAACATGCCGCTTCTGTTCTTC-3 '(Seq ID No: 6)
  • the 5' ends of these two primers contain Mel and BamHI digestion sites, respectively, followed by the 5 'and 3' ends of the target gene, respectively.
  • the sequence, Ndel and BamHI restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET 28b (+) (Novagen, Cat. No. 69865. 3).
  • PCR was performed using the pBS-1659d07 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions are as follows: a total volume of 50 ⁇ 1 contains pBS-1659d07 plasmid 10 P g, primers? 1 '11116:!: —: 3 and? :1: 1.
  • Advantage polymerase Mix (Clontech) 1 ⁇ 1.
  • Cycle parameters 94 ° C 20s, 60 ° C 30s, 68 ° C 2 niin, a total of 25 cycles.
  • Ndel and BamHI The amplified product and the plasmid PET-28 (+) were double-digested, and large fragments were recovered and ligated with T4 ligase.
  • the ligation product was transformed into E. coli DH5a by the calcium chloride method.
  • the bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The affinity chromatography column His. Bind Quick Cartridge (product of Novagen) was used to obtain 6 histidine (6His-Tag). Purified the protein of interest methylation-DM-protein-cysteine methyltransferase 10. After SDS-PAGE electrophoresis, a single band was obtained at 10 kDa ( Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by the Edams hydrolysis method.
  • a peptide synthesizer (product of PE company) was used to synthesize the following methyl-DNA-protein-cysteine methyltransferase 10 specific peptides: '
  • the titer of antibodies in rabbit serum was determined by BLISA using a titer plate coated with 15 ⁇ ⁇ / ⁇ 1 bovine serum albumin peptide complex.
  • Total Ig G was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method proved that the purified antibody could specifically bind to methylated-DNA-protein-cysteine methyltransferase 10.
  • Example 7 Use of a 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.
  • 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 tissues or Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by using a filter hybridization method.
  • 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. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are 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
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other unknown genomic sequences and their complements The region is compared for homology. If the homology with the non-target molecule region is greater than 85% or there are more than 15 consecutive bases, then the primary probe should not be used;
  • Probe l 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 ( pr0 be2), 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 membrane nitrocellulose membrane
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.
  • DNA molecules are required to maintain a high degree of precision and integrity.
  • organisms have evolved a series of repair systems.
  • the main mutagenic and carcinogenic effect of methylation reagents on DNA is the formation of 06-fluorenylguanine.
  • Repair of DNA containing 06-methylguanine is performed by the enzyme 6-0-methylguanine-DNA methyltransferase (methylation-DNA-protein-cysteine methyltransferase) (MGMT) .
  • MGMT methylation-DNA-protein-cysteine methyltransferase
  • Methylation-DNA-protein-cysteine methyltransferase exists in different prokaryotic and eukaryotic organisms, and the area around cysteine residues at the active site of these enzymes is conserved , Can be used as a feature sequence template for this family: [LIVMF]-P- C- H- R- [LIVMF] (2). This specific conserved sequence is required to form its active mot if.
  • the abnormal expression of the characteristic sequence will lead to the loss of the mutant gene repair function of the enzyme, and then cause swelling
  • the novel polypeptide of the present invention has high homology and similarity with methylated-DM-protein-cysteine methyltransferase (MGMT) in structure and function, and the amino acid sequence contains the above-mentioned conservative characteristic sequence
  • the template is considered to belong to the methylation-DNA-protein-cysteine methyltransferase family.
  • the abnormal expression of the above-mentioned specific conserved sequence will cause the function of the polypeptide containing the mot if of the present invention to be abnormal, which will lead to the loss of the function of repairing the mutant gene, and then cause the occurrence of tumor diseases, embryo developmental abnormalities, etc. Including but not limited to: Common tumors in various tissues:
  • Papilloma squamous cell carcinoma [skin, nasopharynx, larynx, cervix], adenoma (carcinoma) [breast, thyroid], mucinous / serous thyroid adenoma (carcinoma) [ovary], basal cell carcinoma [head and facial skin ], (Malignant) polymorphic adenoma [extending gland], papilloma, transitional epithelial cancer [bladder, renal pelvis], etc .;
  • Malignant lymphoma neck, mediastinum, mesenteric and retroperitoneal lymph nodes], various leukemias [lymphoid hematopoietic tissue], multiple myeloma [push / thoracic / rib / skull and long bone], etc .;
  • Nerve fiber [systemic cutaneous nerve / deep nerve and internal organs], (malignant) schwannomas [head, neck, limbs and other nerves], (malignant) glioblastoma [brain], medulloblastoma [ Cerebellum], (malignant) meningiomas [meninges], ganglioblastoma / neuroblastoma [mediastinum and retroperitoneum / adrenal medulla], etc .;
  • malignant melanoma [skin, mucous membrane], (malignant) hydatidiform mole, chorionic epithelial cancer [uterine], (malignant) supporter cells, stromal cell tumor, (malignant) granulosa cell tumor [ovarian, testicular] Blastoma [testis], asexual cell tumor [ovary], embryonal cancer [testis, ovary], (malignant) teratoma [ovary, testis, mediastinum and palate tail], etc .;
  • Cartilage hypoplasia adrenal hypertrophy, microcephaly, no iris, polycystic kidney disease, skin laxity, testicular feminization syndrome, etc .
  • the polypeptide of the present invention and the antagonist, agonist and inhibitor of the polypeptide can be directly used for the treatment of various diseases, such as tumor diseases, embryonic developmental malformations, and the like.
  • the invention also provides screening compounds to identify increased (agonist) or repressed (antagonist) methylation
  • Agonists increase biological functions such as methylation-DNA-protein-cysteine methyltransferase 10 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing methylated-DM-protein-cysteine methyltransferase 10 and labeled methylated-DNA-protein-cysteine can be used in the presence of drugs.
  • Methyltransferases were cultured from 10 to 10. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of methylation-DM-protein-cysteine methyltransferase 10 include alternative antibodies, compounds, receptor deletions, and the like.
  • Methylation-DM-protein-cysteine methyltransferase 10 antagonists can bind to methylation-DNA-protein-cysteine methyltransferase 10 and eliminate its function, or inhibit the polypeptide Production, or binding to the active site of the polypeptide prevents the polypeptide from performing biological functions.
  • methylated-DM-protein-cysteine methyltransferase 10 can be added to the bioanalytical assay, and the methylated-DM-protein-cysteine can be determined by measuring the compound. The effect of the interaction between methyltransferase 10 and its receptor to determine whether a compound is an antagonist. In the same manner as described above for the screening of compounds, it is possible to screen for receptor deletions and analogs that act as antagonists. Polypeptide molecules capable of binding to methylation-DNA-protein-cysteine methyltransferase 10 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, 10 molecules of methylation-DNA-protein-cysteine methyltransferase should be labeled.
  • the present invention provides a method for producing an antibody using a polypeptide, a fragment, a derivative, an analog thereof, or a cell thereof as an antigen.
  • These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the methylation-MA-protein-cysteine methyltransferase 10 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by injecting methylated-DNA-protein-cysteine methyltransferase 10 directly into immunized animals (such as rabbits, mice, rats, etc.), and various adjuvants can be used to enhance Immune response, including but not limited to Freund's adjuvant.
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance Immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing methylated-DNA-protein-cysteine methyltransferase 10 monoclonal antibodies include, but are not limited to, hybridoma technology (Kohler and Miste in. Nature, 1975, 256: 495-497) , Three tumor technology, human B-cell hybridoma technology, EBV-hybridoma technology, etc.
  • the chimeric human antibody constant region and the variable region of non-human origin may be used in combination Pat some production techniques (Morr i son et al, PNAS , 1985, 81: 6851) 0 Only some technical production of single chain antibodies (US Pat No. 4946778) can also be used to produce single-chain antibodies against methylation-DM-protein-cysteine methyltransferase 10.
  • Antibodies Can Be Used In Immunohistochemistry In the biopsy specimen, methylated-DM-protein-cysteine methyltransferase 10 was detected.
  • Monoclonal antibodies that bind to methylated-DM-protein-cysteine methyltransferase 10 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.
  • High affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill methylation-DM-protein-half Cystine methyltransferase 10 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to methylation-DNA-protein-cysteine methyltransferase 10.
  • Administration of an appropriate dose of the antibody can stimulate or block methylation-DNA-protein-cysteine methyltransferase 10 production or activity.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of methylated-MA-protein-cysteine methyltransferase 10 levels.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of methylation-DM-protein-cysteine methyltransferase 10 detected in the test can be used to explain methylation-DNA-protein-cysteine methyltransferase 10 in various diseases The importance and use in the diagnosis of diseases where methylation-DM-protein-cysteine methyltransferase 10 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, and more preferably mass spectrometry.
  • Polynucleotides encoding methylated-DM-protein-cysteine methyltransferase 10 can also be used for a variety of therapeutic purposes.
  • Gene therapy technology can be used to treat abnormal cell proliferation, development, or metabolism caused by non-expression or abnormal / inactive expression of methylation-DM-protein-cysteine methyltransferase 10.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express variant methylation-MA-protein-cysteine methyltransferase 10 to inhibit endogenous methylation-DNA-protein-hemigenesis Cystine methyltransferase 10 activity.
  • a variant methylation-DNA-protein-cysteine methyltransferase 10 may be a shortened methylation-DNA-protein-cysteine methyltransferase lacking a signaling domain Enzyme 10, although it can bind to downstream substrates, lacks signal transduction activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of methylation-DNA-protein-cysteine methyltransferase 10.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • a recombinant polynucleotide encoding methylated-DM-protein-cysteine methyltransferase 10 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 that inhibit methylation-MA-protein-cysteine methyltransferase 10 fflRNA and ribozymes 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 MA to perform endonucleation.
  • Antisense MA, DM and ribozymes can be obtained by any existing RM or DNA synthesis technology, such as the technology of solid phase phosphate amide synthesis of oligonucleotides has been widely used.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RM. This DM sequence has been integrated downstream of the MA polymerase promoter of the vector. In order to increase the stability of the nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • a polynucleotide encoding methylated-DNA-protein-cysteine methyltransferase 10 can be used for the diagnosis of diseases related to methylated-DM-protein-cysteine methyltransferase 10.
  • Polynucleotides encoding methylated-DNA-protein-cysteine methyltransferase 10 can be used to detect the expression of methylated-DNA-protein-cysteine methyltransferase 10 or in disease Aberrant expression of methylated-DM-protein-cysteine methyltransferase 10 in the state.
  • DNA sequences encoding methylated-DNA-protein-cysteine methyltransferase 10 can be used to hybridize biopsy specimens to determine the expression of methylated-DM-protein-cysteine methyltransferase 10 situation.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • Part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a microarray or a DNA chip (also referred to as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in a tissue.
  • Methylation-DM-protein-cysteine methyltransferase 10 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect methylation-DM-protein-cysteine Transcript of acid methyltransferase 10.
  • Methylated-DM-protein-cysteine methyltransferase 10 mutant forms include point mutations compared to normal wild-type methylated-DNA-protein-cysteine methyltransferase 10 DNA sequences, Translocations, deletions, reorganizations, and any other abnormalities. Mutations can be detected using existing techniques such as Southern blotting, MA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • specific sites for each gene on the chromosome need to be identified.
  • only a few chromosome markers based on actual sequence data are available for labeling chromosome positions.
  • an important first step is to locate these DM sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be mapped on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells containing human genes corresponding to the primers will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • oligonucleotide primers of the present invention by a similar method, 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 to 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, for example, in V. Mckusick, Mendel i an Inher i tance 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.
  • the difference in cDNA or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all of the affected individuals and the mutation is not observed in any normal individual, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in the chromosome, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. Based on the resolution capabilities of current physical mapping and gene mapping technology, the CDM that is accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • 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 that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which reminders authorize them to be administered to humans by government agencies that manufacture, use, or sell them.
  • 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.
  • Methylation-DNA-protein-cysteine methyltransferase 10 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of methylated-DNA-protein-cysteine methyltransferase 10 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 diagnostici

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Abstract

L'invention concerne un nouveau polypeptide, une protéine de méthylation d'ADN-cystéine méthyl-transférase 10, et un polynucléotide codant 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 de tumeurs et d'anomalies apparaissant lors du développement de l'embryon. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la protéine de méthylation d'ADN-cystéine méthyl-transférase 10.
PCT/CN2001/000943 2000-06-12 2001-06-11 Nouveau polypeptide, proteine de methylation d'adn-cysteine methyl-transferase 10, et polynucleotide codant ce polypeptide WO2002000827A2 (fr)

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CN00116428A CN1328144A (zh) 2000-06-12 2000-06-12 一种新的多肽——甲基化－dna－蛋白－半胱氨酸甲基转移酶10和编码这种多肽的多核苷酸
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