WO2002000826A2 - Nouveau polypeptide, proteine humaine de methylation d'adn, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine de methylation d'adn, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2002000826A2
WO2002000826A2 PCT/CN2001/000942 CN0100942W WO0200826A2 WO 2002000826 A2 WO2002000826 A2 WO 2002000826A2 CN 0100942 W CN0100942 W CN 0100942W WO 0200826 A2 WO0200826 A2 WO 0200826A2
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polypeptide
protein
polynucleotide
dna
cysteine methyltransferase
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PCT/CN2001/000942
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Chinese (zh)
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WO2002000826A3 (fr
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Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU89532/01A priority Critical patent/AU8953201A/en
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Publication of WO2002000826A3 publication Critical patent/WO2002000826A3/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.)

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, namely human methylation-DNA-protein-cysteine methyltransferase 1 3, and a polynucleotide encoding the polypeptide. sequence. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. 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 DNA is the formation of 06-fluorenylguanine.
  • Repair of DM containing 06-alkylguanine is performed by the enzyme 6-0-methylguanine-DNA methyltransferase (methylation-DNA-protein-cysteine methyltransferase) (MGMT) .
  • MGMT methylation-DNA-protein-cysteine methyltransferase
  • the alkyl group at position 0-6 is transferred to one cysteine residue in the enzyme (Lindahl T., Sedgwick B., Sek i guchi M., Nakabeppu ⁇ ., Annu. Rev. B i ochem 57: 1 33-157 (1 988)).
  • This is a suicide reaction: the enzyme is irreversibly inactivated, and each methyl group removed from a DNA molecule consumes a new enzyme molecule, and methylated proteins accumulate as a dead-end product.
  • 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]-P- C- H- R- [LIVMF] (2).
  • point mutations such as ra s, p53, and ERCC-3 genes. These mutations are extremely important for activating (oncogenes) or suppressing (tumor suppressor genes) these genes.
  • Point mutations caused by amidines (such as N-nitroso compounds) on DM are involved in triggering specific humans cancer.
  • MGMT can specifically repair DNA mutagenic damage caused by alkylating agents, 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 (L indah l et al, Annu. Rev. Biochem. (1988) 57, 133-157; AE Pegg, Cancer Res. (1990) 50, 6119-6129).
  • novel human methylated-DNA-protein-cysteine methyltransferase and methylated-DNA-protein-cysteine methyltransferase are 81% identical at the protein level and 91 % Similarity, and also contains a methylated-DNA-protein-cysteine methyltransferase family member characteristic sequence template, and has similar biological functions, so it is considered to belong to methylated-DNA- Protein-cysteine methyltransferase family, Designated as human methylation-DNA-protein-cysteine methyltransferase 13.
  • 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, Cawkwell L., Potter PM, Margison GP, Nucleic Acids Res 20: 1891-1895 (1992)).
  • the human methylation-DM-protein-cysteine methyltransferase 13 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, Therefore, there is always a need in the art to identify more human methylation-DNA-protein-cysteine methyltransferase 13 proteins involved in these processes, especially the amino acid sequence of such proteins.
  • the new human methylation-DNA-protein-cysteine methyltransferase 13 protein gene isolation 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 the disease, so it is important to isolate its code for DM. Disclosure of 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 human methylated-DNA-protein-cysteine methyltransferase 13.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding human methylation-DNA-protein-cysteine methyltransferase 13.
  • Another object of the present invention is to provide a method for producing human methylated-DM-protein-cysteine methyltransferase 13.
  • Another object of the present invention is to provide an antibody against the human methylation-DNA-protein-cysteine methyltransferase 13 of the polypeptide of the present invention.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the human methylation-DM-protein-cysteine methyltransferase 13 of the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities in human methylation-DM-protein-cysteine methyltransferase 13.
  • the present invention relates to an isolated polypeptide, which is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the present invention also relates to an isolated polynucleotide comprising a nucleotide sequence selected from the group consisting of: Its variant:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) having SEQ ID NO: 1
  • the present invention further relates to a vector, particularly an expression vector, containing the polynucleotide of the present invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit human methylation-DM-protein-cysteine methyltransferase 13 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 human methylation-DNA-protein-cysteine methyltransferase 13 protein in vitro, which comprises detecting the polypeptide or a susceptibility in a biological sample A mutation in a coding 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 comprising a polypeptide of the present invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the invention also relates to the polypeptides and / or polynucleotides of the invention in the preparation for the treatment of cancer, developmental or immune diseases or other due to human methylation-DNA-protein-cysteine methyltransferase 1 3 Use of medicines for diseases caused by abnormal expression.
  • 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 DM or RM, 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 acids or nucleotide changes, or a polynucleotide sequence encoding it.
  • the changes may include amino acid sequences or nucleotides A deletion, insertion, or substitution of an amino acid or nucleotide in a sequence.
  • Variants may have "conservative" changes in which the substituted amino acid 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 refers to an alteration in the amino acid sequence or nucleotide sequence that results in an increase in one or more amino acids or nucleotides compared to a naturally occurring molecule.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • 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 in appropriate animals or cells and to bind to specific antibodies.
  • An "agonist” refers to a molecule that, when combined with human methylation-DNA-protein-cysteine methyltransferase 13, can cause the protein to change, thereby regulating the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind human methylation-DNA-protein-cysteine methyltransferase 13.
  • Antagonist refers to a type that can block or regulate human methylation-DM-protein- when combined with human methylation-DNA-protein-cysteine methyltransferase 13 Cysteine methyltransferase 13 is a biologically or immunologically active molecule. Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates, or any other molecule that can bind human methylation-DNA-protein-cysteine methyltransferase 13.
  • Regular refers to changes in the function of human methylation-DM-protein-cysteine methyltransferase 13, including increased or decreased protein activity, changes in binding characteristics, and human methylation-DNA- Alteration of any other biological, functional or immune properties of protein-cysteine methyltransferase 13.
  • Substantially pure ' means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human methylation-DM-protein- using standard protein purification techniques. Cysteine methyltransferase 1 3.
  • Essentially pure human methylation-DNA-protein-cysteine methyltransferase 1 3 produces a single main band on a non-reducing polyacrylamide gel
  • the purity of human methylated-DNA-protein-cysteine methyltransferase 13 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to a polynucleotide that naturally binds by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence C-T-GA
  • complementary sequence G-A-C-T
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • Homology refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid.
  • the inhibition of such hybridization can be detected by performing hybridization (Southern blotting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are 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 number of matching residues between sequence A and sequence X 100 The number of residues in sequence A-the number of spacer residues in sequence A The number of spacer residues in a sequence B can also be determined by Clus ter method or using methods known in the art such as Jotim Hein.
  • the percent identity between nucleic acid sequences He in J "(1990) Methods in emzumology 183: 625-645) 0 "Similarity” refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions when the alignment between amino acid sequences is aligned.
  • negatively charged amino acids may include aspartic acid and glutamic acid.
  • Amino acids; positively charged amino acids may include lysine and arginine; amino acids with similarly charged head groups that have similar hydrophilicity 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 specific DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? , which can be specific sexually binds human methylated-DNA-protein-cysteine methyltransferase 13 epitopes.
  • 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 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 certain vector, or such a polynucleotide or polypeptide may be part of a certain composition. Since the carrier or composition is not part 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 methylation-DNA-protein-cysteine methyltransferase 1 3 refers to human methylation-DNA-protein-cysteine methyltransferase 1 3 basically Contains no other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify human methylated-DNA-protein-cysteine methyltransferase 1 3 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human methylated-DNA-protein-cysteine methyltransferase 1 3 peptides can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human methylation-DNA-protein-cysteine methyltransferase 13, 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, yeasts, higher plants, insects, and mammalian cells) using recombinant techniques.
  • polypeptides of the invention may be glycosylated, or 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 human methylation-DNA-protein-cysteine methyltransferase 1 3.
  • fragment As used in the present invention, the terms “fragment”, “derivative” and “analog” refer to the biologically-maintained human methylation-DNA-protein-cysteine methyltransferase 1 3 of the present invention. Functional or active polypeptide.
  • 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 conserved or non-conserved (preferably conservative) Amino acid residues), and the substituted 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 A substituent; or (II) a method in which a mature polypeptide is fused with another compound (such as a compound that extends the half-life of a polypeptide, such as polyethylene glycol); or (IV) a method in which an additional amino acid sequence is fused Polypeptide sequences (such as leader sequences or secreted sequences or sequences used to purify this polypeptide or protease sequences) formed from mature polypeptides are described herein. Such fragments, derivatives and analogs are considered to be within the skill of those skilled in the art Within knowledge.
  • 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 1964 bases, and its open reading frame of 299-661 encodes 120 amino acids.
  • this polypeptide has 81% homology with human methylation-DNA-protein-cysteine methyltransferase, and it can be deduced that the human methylation-DNA-protein- Cysteine methyltransferase 13 has a similar structure and function to human methylation-DNA-protein-cysteine methyltransferase.
  • the polynucleotide of the present invention may be in the form of DNA or RM.
  • 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 having a sequence different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • 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 present invention also relates to a polynucleotide that hybridizes to a sequence described above 50% less, preferably 70% identity).
  • the invention particularly relates to polynucleotides that can hybridize 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, 60 ° C; or (2) added during hybridization Use a denaturant, such as 50 ° /.
  • 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 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 human methylated-DNA-protein-cysteine methyltransferase 13.
  • 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 methylation-DNA-protein-cysteine methyltransferase 13 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 DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of MA sequences is often the method of choice.
  • the more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating 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.
  • the construction of cDNA libraries is also a common method (Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989).
  • Commercially available cDNA libraries are also available, such as different cDNA libraries from Cloiitech. 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 DM-RNA hybridization; (2) the presence or absence of marker gene functions; (3) determination of human methylation-DNA-protein-cysteine alpha Levels of transcripts of glycosyltransferase 13; (4) Detecting gene-expressed protein products by immunological techniques or by measuring biological activity. The above method can be used alone or Multiple methods can be used 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 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 human methylation-DM-protein-cysteine methyltransferase 13 gene expression can be detected using immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked Immunoadsorption (ELISA) and so on.
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked Immunoadsorption (ELISA) and so on.
  • a method using PCR technology to amplify DNA / RNA is preferably used to obtain the gene of the present month.
  • 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 DM / RNA fragment 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 determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the 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 human methylation-DNA-protein-cysteine methyltransferase 13 coding sequence. , And a method for producing the polypeptide of the present invention by recombinant technology.
  • a polynucleotide sequence encoding human methylation-DNA-protein-cysteine methyltransferase 13 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 origins of replication, promoters, marker genes, and translation regulators. Controlling components.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing DM sequences encoding human methylation-DNA-protein-cysteine methyltransferase 13 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology (Sambroook, et al. Molecuar ar Cl on ing, a Labora tory Manua l, cold Spring Harbor Labora tory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the l ac or trp promoter of E.
  • coli the PL promoter of lambda phage
  • eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, and the early and late SV40 promoters Promoters, retroviral LTRs, and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • Expression vectors also include ribosome binding sites and transcription terminators for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells.
  • Enhancers are cis-acting factors expressed by DM, usually about 10 to 300 base pairs, which act on the promoter to enhance gene transcription. Examples include SV40 enhancer of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancer and adenovirus enhancer on the late side of the origin of replication.
  • 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 methylation-DNA-protein-cysteine methyltransferase 13 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute the polynucleus.
  • the term "host cell” refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S2 or Sf 9 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 DNA uptake can be harvested after exponential growth phase, with (: Treatment 1 2, steps well known in the art with alternative is MgC l 2
  • the transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, Or conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human methylation-DNA-protein-cysteine methyltransferase 1 3 (Scence, 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.
  • recombinant proteins can be separated and purified by various separation methods using their physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • 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
  • FIG. 1 is a comparison diagram of amino acid sequence homology of human methylation-DM-protein-cysteine methyltransferase 13 and human methylation-DNA-protein-cysteine methyltransferase of the present invention .
  • the upper sequence is human methylation-DNA-protein-cysteine methyltransferase 1 3
  • the lower sequence is human methylation-DNA-protein-cysteine methyltransferase.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+".
  • FIG. 2 is a polyacrylamide gel electrophoresis image (SDS-PAGE) of isolated human methylated-DM-protein-cysteine methyltransferase 1 3.
  • 1 3kDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • RNA Human fetal brain total RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RM using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA. Use Smart cDNA Cloning Kit (purchased from Clontech). The 0 fragment was inserted into the multicloning site of pBSK (+) vector (Clontech), and transformed into DH5 ⁇ . The bacteria formed a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones 1614 ⁇ 2 was new DNA.
  • the inserted cDNA fragments contained in this clone were determined in both directions by synthesizing a series of primers.
  • the sequence of the human methylation-DM-protein-cysteine methyltransferase 13 and its encoded protein sequence of the present invention was performed using the Blas t program (Basicloca l Al ignment search tool) [Al tschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], homology search was performed in Genbank, Swissport and other databases.
  • the gene most homologous to the human methylation-DNA-protein-cysteine methyltransferase 13 of the present invention is a known human methylation-DNA-protein-cysteine methyltransferase Enzyme, its accession number is D31763 in Genbank.
  • the protein homology results are shown in Figure 1. The two are highly homologous, with 81% identity; 91% similarity.
  • Example 3 Cloning of a gene encoding human methylation-DNA-protein-cysteine methyltransferase 13 by RT-PCR
  • CDNA was synthesized using fetal brain total RNA as a template and ol igo-dT as a primer for reverse transcription reaction. After purification of Qiagene's kit, PCR amplification was performed with the following primers:
  • Pr imer l 5, one GGGGCATGTCTGCCATATGGCTCC —3, (SEQ ID NO: 3)
  • Pr imer2 5'- CATAGGCCGAGGCGGCCGACATGT -3, (SEQ ID NO: 4)
  • Pr imerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp;
  • Pr imer2 is the 3'-end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 ⁇ l / L KC1, 10 ⁇ l / L Tris-Cl, (pH 8.5.5), 1.5 mmol / L MgCl 2 , 200 ⁇ mol in a reaction volume of 50 ⁇ 1 / L dNTP, l Opmol primer, 1U Taq DM polymerase (Clontech company product).
  • 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 0
  • ⁇ -act in was set as positive during RT-PCR Controls and template blanks are negative controls.
  • the amplified product was purified using a QIAGEN kit and ligated to a pCR vector (Invitrogen) using a TA cloning kit.
  • the DNA sequence analysis results showed that the DM sequence of the PCR product was exactly the same as shown in SEQ ID NO: 1:!-1964bp '.
  • Example 4 Analysis of human methylation-DNA-protein-cysteine methyltransferase 13 gene expression by Northern blot method:
  • RNA was synthesized by electrophoresis on a 1.2% agarose gel containing 20 ⁇ lM of 3- (N-morpholino) propanesulfonic acid (pH 7.0) -5 ⁇ m sodium acetate-ImM EDTA-2.2M formaldehyde. It was then transferred to a nitrocellulose membrane.
  • 32 P dATP Preparation 32 ⁇ - DNA probe labeled by the random primer Method - with cc.
  • the DNA probe used was the human methylated-DNA-protein-cysteine methyltransferase 13 coding region sequence (299bp to 661bp) amplified by PCR as shown in FIG.
  • Primer 3 5'-CATGCTAGCATGATCACATCCCAGGGATCAGTG-3 '(Seq ID No: 5)
  • Primer4 5'- CATGGATCCCTAGCATTGGCCCCCTGGTCCCAC- 3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Nhel and BamHI restriction sites, respectively , followeded by the coding sequences of the 5 'end and 3' end of the gene of interest, respectively.
  • the Nhel and BaraHI restriction sites correspond to the selectivity on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Endonuclease site.
  • the PCR reaction was performed using the pBS-1614 ⁇ 2 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: pBS-] in a total volume of 50 ⁇ 1; 10] 4f 12 plasmid 10 pg, primers Primer-3 and Primer-4. J is lpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94. C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles. Nhel 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 E. coli DH5a by the calcium chloride method. After being cultured overnight in LB plates containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1), positive clones were selected by colony PCR method and sequenced. A positive clone ( ⁇ ET-1614 ⁇ 2) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) using the calcium chloride method. In LB liquid medium containing kanamycin (final concentration 30 ⁇ g / ml), the host strain BL21 ( ⁇ -1614 ⁇ 2) was at 37. C.
  • NH2-Met-Ile-Thr-Ser-Gln-Gly-Ser-Val-Ser-Phe-Arg-Asp-Val-Thr-Val-C00H (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For the method, see: Avrameas, et al. I see unochemistry, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Total IgG was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and the total IgG Anti-peptide antibodies were isolated.
  • the immunoprecipitation method proved that the purified antibody could specifically bind to human methylated-DNA-protein-cysteine methyltransferase 13.
  • 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 tissue or pathology. Whether the expression in tissue cells is abnormal.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method Acid sequence or a homologous polynucleotide sequence thereof.
  • Filter hybridization methods include dot blotting, Sou thern imprinting, 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 to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature), so that the hybridization background is reduced and only strong specific signals are retained.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • 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 known genomic sequences and their complements Region for homology comparison, if the homology with non-target molecular region is greater than 85% After 15 consecutive bases are identical, the primary probe should not be used in general;
  • 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 (probe2), which belongs to the second type of probe, is equivalent to the replacement mutant sequence of the gene fragment of SEQ'ID NO: 1 or its complementary fragment (41Nt):
  • 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
  • probe 1 can be used to qualitatively and quantitatively analyze the presence and differential expression of the polynucleotide of the present invention in different tissues.
  • 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 accuracy 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 A containing 06-alkylguanine is performed by the enzyme 6-0-methylguanine-DNA methyltransferase (methylation-DNA-protein-cysteine methyltransferase) (MGMT) .
  • MGMT methylation-DNA-protein-cysteine methyltransferase
  • MGMT methylation-DNA-protein-cysteine methyltransferase
  • Methylation-DNA-protein-cysteine methyltransferase exists in different prokaryotic and eukaryotic organisms, and its abnormal expression in the body can cause the body to lose the repair function of mutant genes, and further Causes the occurrence of related diseases.
  • the polypeptide of the present invention has 81% identity and 91% similarity with human methylation-DNA-protein-cysteine methyltransferase at the protein level, and also contains methylation-DNA-protein- Characteristic sequence template shared by members of the cysteine methyltransferase family and has similar biological functions, so it is considered to belong to the methylation-DNA-protein-cysteine methyltransferase family.
  • This polypeptide is in vivo Abnormal expression can affect the body's repair of mutant genes, and then lead to the occurrence of tumor diseases and embryo developmental malformations, these diseases include but are not limited to: Common tumors in various tissues:
  • Papilloma squamous cell carcinoma [skin, nasopharynx, larynx, cervix], adenoma (carcinoma) [breast, thyroid], mucinous / serous cystadenomas (carcinoma) [ovary], basal cell carcinoma [head and face Skin], (malignant) polytype adenoma [extending gland], papilloma, transitional epithelial cancer [bladder, spleen], etc .; 2.
  • sarcoma Fibrous (sarcoma) [limbs], (Malignant) fibrohistiocytoma [limbs], lipo (sarcoma) [subcutaneous tissue, lower limbs, retroperitoneum], leiomyosarcoma (uterus and gastrointestinal), striated muscle (Sarcoma) [head and neck, genitourinary tract, limbs], hemangio (sarcoma), lymphangioma (sarcoma) [skin, subcutaneous tissue, tongue, lips], bone (sarcoma) [cranium, long bone], (malignant Giant cell tumor [femoral / tibia / upper humerus], cartilage (sarcoma) [hand and foot short bone, pelvis / rib / femoral / humerus / scapula], synovial (sarcoma) tumor [knee / ankle / wrist / shoulder / Near the elbow], (malignant) mesotheli
  • 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) schwannoma [nervous of head, neck, limbs, etc.], (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] fine Blastoma [testis], asexual cell tumor [ovary], embryonal cancer [testis, ovary], (malignant) teratoma [ovary, testis, mediastinum and palate tail], etc .
  • malignant melanoma skin, mucous membrane
  • hydatidiform mole chorionic epithelial cancer [uterine]
  • (malignant) supporter cells stromal cell tumor
  • (malignant) granulosa cell tumor ovarian, testicular] fine Blastoma [testis]
  • asexual cell tumor ovary
  • embryonal cancer testis, ovary
  • (malignant) teratoma
  • Cartilage hypoplasia adrenal hypertrophy, microcephaly, no iris, polycystic kidney disease, skin relaxation, testicular feminization syndrome, etc.
  • the polypeptide of the present invention and its antagonist, agonist and inhibitor can be directly It is used for the treatment of a variety of diseases, such as tumor diseases, embryonic developmental malformations, and the like.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human methylation-DNA-protein-cysteine methyltransferase 13.
  • Agonists increase human methylation-DNA-protein-cysteine methyltransferase 13 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing human methylation-DNA-protein-cysteine methyltransferase 13 and a labeled human methylation-DM-protein-cysteine can be prepared in the presence of a drug.
  • the amino acid methyltransferase 13 is cultured together.
  • Antagonists of human methylation-DNA-protein-cysteine glutamyl transferase 13 include antibodies, compounds, receptor deletions, and the like that have been screened.
  • An antagonist of human methylation-DNA-protein-cysteine methyltransferase 1 3 can bind to human methylation-DNA-protein-cysteine methyltransferase 1 3 and eliminate its function, or It is to inhibit the production of the polypeptide or to bind to the active site of the polypeptide so that the polypeptide cannot perform a biological function.
  • human methylation-DNA-protein-cysteine methyltransferase 13 can be added to the bioanalytical assay, and the human methylation-DNA-protein-hemi- Influence of the interaction between cystine methyltransferase 13 and its receptor to determine whether a compound is an antagonist.
  • Receptor deletions and analogs that act as antagonists can be screened in the same way as above for screening compounds.
  • Polypeptide molecules capable of binding to human methylated-DNA-protein-cysteine methyltransferase 13 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, human methylation-DNA-protein-cysteine methyltransferase 13 molecules should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against the human methylation-DNA-protein-cysteine methyltransferase 13 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 human methylation-MA-protein-cysteine methyltransferase 1 3 direct injection in immunized animals (such as rabbits, mice, rats, etc.), a variety of adjuvants are available For enhancing the immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies to human methylated-DNA-protein-cysteine methyltransferase 1 3 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.
  • Anti-human methylation-DM-protein-cysteine methyltransferase 13 antibody can be used in immunohistochemistry to detect human methylation-DNA-protein-cysteine methyltransferase in biopsy specimens Enzyme 1 3.
  • Monoclonal antibodies that bind to human methylated-DM-protein-cysteine methyltransferase 13 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 methylated-DNA-egg White-cysteine methyltransferase 13 High-affinity monoclonal antibodies can covalently bind to bacterial or plant toxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human methylation-DNA-protein- Cysteine methyltransferase 13 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human methylation-DM-protein-cysteine methyltransferase 13.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human methylated-DNA-protein-cysteine methyltransferase 13.
  • the invention also relates to a diagnostic test method for quantitatively and locally detecting the level of human methylation-DNA-protein-cysteine methyltransferase 1 3.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the levels of human methylation-DM-protein-cysteine methyltransferase 13 detected in the test can be used to explain human methylation-DNA-protein-cysteine methyltransferase 1 3 in each The importance of these two diseases and for the diagnosis of diseases where human methylation-DNA-protein-cysteine methyltransferase 13 plays a role.
  • 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 analysis.
  • the polynucleotide encoding human methylated-DM-protein-cysteine methyltransferase 13 can also be used for a variety of therapeutic purposes.
  • Gene therapy technology can be used to treat abnormal cell proliferation, development or metabolism caused by the non-expression or abnormal / inactive expression of human methylation-DM-protein-cysteine methyltransferase 1 3.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human methylation-DNA-protein-cysteine methyltransferase 13 to inhibit endogenous human methylation-DM-protein -Cysteine methyltransferase 1 3 activity.
  • a mutant human methylation-DNA-protein-cysteine methyltransferase 1 3 may be a shortened human methylation-DNA-protein-cysteine without a signaling domain.
  • Methyltransferase 13 although it can bind to downstream substrates, lacks signaling activity. Therefore, the recombinant gene therapy vector can be used for treating diseases caused by abnormal expression or activity of human methylation-DM-protein-cysteine methyltransferase 13.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • a recombinant viral vector carrying a polynucleotide encoding human methylation-DNA-protein-cysteine methyltransferase 13 can be found in the existing literature (Sarabrook, et al.).
  • a recombinant polynucleotide encoding human adenylated-DNA-protein-cysteine methyltransferase 13 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: injecting the polynucleotide directly into a tissue in vivo; or introducing the polynucleotide into a cell through a vector (such as a virus, phage, or plasmid) in vitro, The cells are then transplanted into the body and the like.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit human methylation-DM-protein-cysteine methyltransferase 1 3 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 MA to perform endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DM synthesis techniques, 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 a DNA sequence encoding the RM. This DM sequence has been integrated downstream of the vector's RNA polymerase 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 linkage between ribonucleosides using phosphothioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human methylation-DM-protein-cysteine methyltransferase 13 can be used for diseases related to human methylation-DNA-protein-cysteine methyltransferase 13 diagnosis.
  • a polynucleotide encoding human methylation-DNA-protein-cysteine methyltransferase 1 3 can be used to detect the expression of human methylation-DM-protein-cysteine methyltransferase 1 3 Or the abnormal expression of human methylation-DM-protein-cysteine methyltransferase 1 3 in a disease state.
  • the DNA sequence encoding human methylation-DM-protein-cysteine methyltransferase 1 3 can be used to hybridize biopsy specimens to determine human methylation-DNA-protein-cysteine methyltransferase. 1 3 expression status.
  • Hybridization techniques include Sou thern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are all mature and open technologies, and related kits are commercially available.
  • Part or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray (Microray) or a DM chip (also known as a "gene chip") for analyzing differential expression analysis of genes in tissues and Genetic diagnosis.
  • Human methylation-DNA-protein-cysteine methyltransferase 13 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect human methylation-DNA-protein-hemi Transcription product of cystine methyltransferase 1 3.
  • Human methylated-DM-protein-cysteine methyltransferase 1 3 mutant forms include comparisons to normal wild-type human methylated-DNA-protein-cysteine methyltransferase 1 3 DNA sequences Point mutations, translocations, deletions, recombinations and any other abnormalities. Mutations can be detected using well-known techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • the specificity of each gene on the chromosome needs to be identified Site.
  • an important first step is to locate these DM sequences on a chromosome.
  • a PCR primer (preferably 15-35bp) is prepared from the cDNA, and the sequence can be located on the chromosome. 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, for example, V. Mckusick, Mendel i aii Inher i tance in Man (available online with Johns Hopk ins Un ivers i ty We l ch Med i ca l Li brary). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the difference in cDM or genomic sequence between the affected and unaffected individuals needs to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the CDM that is accurately mapped to a disease-related chromosomal region 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 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.
  • containers there can be medicines manufactured, used or sold by Instructions given by the government regulatory agency for the product or biological product, which reflects the permission of the government regulatory agency for production, use, or sale to be administered to the human body.
  • 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 methylation-DNA-protein-cysteine methyltransferase 1 3 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and dose range of human methylated-DNA-protein-cysteine methyltransferase 13 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 .

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Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine de méthylation d'ADN, 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 de malformations 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 humaine de méthylation d'ADN.
PCT/CN2001/000942 2000-06-12 2001-06-11 Nouveau polypeptide, proteine humaine de methylation d'adn, et polynucleotide codant ce polypeptide WO2002000826A2 (fr)

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US8017636B2 (en) 2003-04-11 2011-09-13 Ptc Therapeutics, Inc. 1,2,4-Oxadiazole benzoic acid compositions and their use in bioassays
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US8299105B2 (en) 2003-04-11 2012-10-30 Ptc Therapeutics, Inc. 1,2,4-oxadiazole benzoic acid compositions and their use in bioassays
US8486982B2 (en) 2003-04-11 2013-07-16 Ptc Therapeutics, Inc. 1,2,4-oxadiazole benzoic acids
US8975287B2 (en) 2003-04-11 2015-03-10 Ptc Therapeutics, Inc. Methods for using 1,2,4-Oxadiazole benzoic acid compounds
CN114916502A (zh) * 2022-07-07 2022-08-19 电子科技大学 一种视网膜色素变性疾病模型的构建方法和应用
CN114916502B (zh) * 2022-07-07 2023-06-16 电子科技大学 一种视网膜色素变性疾病模型的构建方法和应用

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