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WO2001070960A1 - Nouveau polypeptide, proteine humaine de reparation 13 du mesappariement de l'adn, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine de reparation 13 du mesappariement de l'adn, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001070960A1
WO2001070960A1 PCT/CN2001/000369 CN0100369W WO0170960A1 WO 2001070960 A1 WO2001070960 A1 WO 2001070960A1 CN 0100369 W CN0100369 W CN 0100369W WO 0170960 A1 WO0170960 A1 WO 0170960A1
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Prior art keywords
polypeptide
polynucleotide
mismatch repair
repair gene
gene protein
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc
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Shanghai Biowindow Gene Development Inc
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Priority to AU48244/01A priority Critical patent/AU4824401A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a novel polypeptide __ human DNA mismatch repair gene protein 13, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and the polypeptide. Background technique
  • This repair system consists of a variety of mut genes (such as MutH, MutU MutS, and MutU) and various single-stranded DNA binding proteins (such as Exonuclease I, exonuclease VII, RecJ exonuclease, DNA polymerase III holoenzyme and DNA ligase) are jointly completed.
  • mut genes such as MutH, MutU MutS, and MutU
  • DNA binding proteins such as Exonuclease I, exonuclease VII, RecJ exonuclease, DNA polymerase III holoenzyme and DNA ligase
  • MutS protein is responsible for identifying and combining with mismatched bases in this process to form MutS-DNA complexes
  • MutL interacts with MutS-DNA complexes
  • MutH binds to unmethylated sites on the hemimethylated GATC site to catalyze the repair of DNA mismatched bases.
  • MutS a DNA mismatch repair gene
  • DNA mismatch repair system is also highly conservative in evolution.
  • MutS protein superfamily a large protein family, namely the MutS protein superfamily.
  • Reenan et al. Cloned various MutS homologous proteins from Saccharomyces cerevisiae.
  • MutS protein homologous proteins MSH in Saccharomyces cerevisiae.
  • MSH proteins are different in different organisms. The repair of DNA mismatched bases is involved in tissues, and its DNA repair mechanism is much more complicated than that in prokaryotes.
  • the human DM mismatch repair gene protein 13 protein plays an important role in regulating important functions of the body such as cell division and embryonic growth, and it is believed that a large number of eggs are involved in these regulatory processes, so identification in the art has always been required. More human DNA mismatch repair gene protein 13 proteins involved in these processes, especially the amino acid sequence identification of this protein.
  • Newcomer DM Mismatch Repair Gene Protein 1 3 Protein Isolation The coding gene also provides £ 5 for research to determine its role in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for diseases, so isolation of its compiled DNA is very important. Disclosure of invention
  • An object of the present invention is to provide an isolated novel polypeptide-human DNA mismatch repair gene egg 3 1 3 and fragments, analogs and derivatives thereof.
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human DNA mismatch repair gene protein 1 3.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human DM mismatch repair gene protein 1 3.
  • Another object of the present invention is to provide a method for producing a human DNA mismatch repair gene protein 1 3.
  • Another object of the present invention is to provide a human DNA mismatch repair gene for a polypeptide of the present invention.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors of the polypeptide-to-human DNA mismatch repair gene protein 13 of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to the abnormality of human DNA mismatch repair gene protein 1 3.
  • the present invention relates to an isolated polypeptide, which is of human origin, and includes: a polypeptide having the amino acid sequence of SEQ ID D. 2, or a conservative variant, biologically active fragment, or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 1 366-1725 in SEQ ID NO: 1; and (b) having a sequence 1- in SEQ ID NO: 1 2345-bit sequence.
  • 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 the activity of human DNA mismatch repair gene protein 13 protein, which comprises using 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 DNA mismatch repair gene protein 1 3 protein in vitro, which comprises detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, Alternatively, the amount or biological activity of a polypeptide of the invention in a biological sample is detected.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the preparation of the polypeptide and / or polynucleotide of the present invention for the treatment of malignant tumors, hematological diseases, developmental disorders, HIV infection and immune diseases and various types of inflammation or other gene proteins due to human DNA mismatch repair. 13. Use of a medicine 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 DNA or RNA, which can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to oligopeptides, peptides, polypeptides, or protein sequences and fragments or points 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 the complete natural related to the protein-molecule. Amino acid.
  • a “variant" of a protein or polynucleotide refers to an amino acid sequence having one or more amino acids or nucleotide changes or a polynucleotide sequence encoding it.
  • the changes may include deletions, insertions or substitutions of amino acids or nucleotides in the amino acid sequence or nucleotide sequence.
  • Variants can have "conservative" changes, in which the amino acid substituted has a structural or chemical property similar to the original amino acid, such as replacing isoleucine with leucine.
  • Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion 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 proteins with the structural, regulatory, or biochemical functions of natural molecules.
  • immunological activity refers to natural, recombinant, or synthetic proteins and fragments thereof that induce specific Immune response and ability to bind specific antibodies.
  • An "agonist” refers to a molecule that, when combined with human DNA mismatch repair gene protein 1 3, can cause the protein to change and thereby regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that can bind to human DNA mismatch repair gene protein 13.
  • Antagonist refers to a molecule that can block or regulate the biological or immunological activity of human DNA mismatch repair gene protein 13 when combined with human DNA mismatch repair gene protein 13.
  • Antagonists and inhibitors can include proteins, nucleic acids, carbohydrates or any other molecule that can bind to human DNA mismatch repair gene protein 13.
  • Regular refers to a change in the function of human DNA mismatch repair gene protein 1 3, including the increase or decrease of protein activity, changes in binding characteristics, and any other organism of human DNA mismatch repair gene protein 1 3 Changes in nature, function, or immunity.
  • substantially pure ' means essentially free of other proteins, lipids, sugars or other substances naturally associated with it.
  • Those skilled in the art can use standard protein purification techniques to purify human DNA mismatch repair gene protein 1 3, : The substantially pure human DNA mismatch repair gene protein 13 produces a single main band on a non-reducing polyacrylamide gel. The purity of human DNA mismatch repair gene protein 13 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence "C-T-G-A” may be combined with the complementary sequence "G-A-C-T", and 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 imprinting or Northern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that conditions with reduced stringency allow non-specific binding, because conditions with reduced stringency require that the two sequences bind to each other as either specific or selective interactions.
  • 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 percentage identity can be determined electronically, such as by the MEGALIGN program (Lasergene software 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 Cluster method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups.
  • the two amino acid sequences are as follows: ⁇ ) The percent identity between A and B is calculated by the following formula: Number of residues matching between sequence A and sequence X 100 Number of residues in sequence A-interval residues in sequence A The number of spacer residues in radix-sequence B can also be determined by the Cluster method or by methods known in the art such as Jotun He in (%). 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 may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to the "sense strand”.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification ⁇ : ⁇ replaces 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. F (ab ') 2 and Fv, which can heterogeneously bind to the epitope of human DNA mismatch repair gene protein 13.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen-binding region has been 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 vector, or such a polynucleotide or polypeptide may be part of a 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 the natural state in living cells 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 DNA mismatch repair gene protein 1 3 refers to human DNA mismatch ⁇ complex gene protein 1 3 is substantially free of other proteins, lipids, carbohydrates or other substances naturally associated with it. This Those skilled in the art can use standard protein purification techniques to purify human DM mismatch repair gene protein 1 3. A substantially pure polypeptide can produce a single main band on a non-reducing polyacrylamide gel. The purity of human DNA error repair protein 1 3 peptides can be analyzed by amino acid sequence.
  • the present invention provides a novel polypeptide-to-human DNA mismatch repair gene protein 1 3, which is basically composed of
  • 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. Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated.
  • the polypeptides of the present invention may also include or exclude the initial methiothione, acid residues.
  • the invention also includes fragments, derivatives, and the like of human DM mismatch repair gene protein 1 3 :
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human DNA mismatch repair gene protein 1 3 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: U) a type in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substituted
  • the amino acid may or may not be encoded by the genetic code; or ( ⁇ ) such that a group on one or more amino acid residues is substituted by another group to include a substituent; or ( ⁇ ⁇ ) like this
  • a type 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
  • an additional amino acid sequence is fused into a mature polypeptide Sequences (such as leader sequences or secretory sequences or sequences used to purify this polypeptide or protease sequences)
  • sequences such as leader sequences or secretory sequences or sequences used to purify this polypeptide or protease sequences
  • 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 2 345 bases, and its open reading frame 1 366-1 725 encodes 119 amino acids.
  • this peptide has a similar expression profile with the human DNA mismatch repair gene protein MSH 3, and it can be inferred that the human DNA mismatch repair gene protein 1 3 has a human DNA mismatch repair gene protein MSH 3 Functions.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 in the present invention, but which differs from the coding region sequence shown in SEQ ID NO: 1.
  • 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 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 may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants PT / CNO 1/00369 body, deletion variant, and insertion variant.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity, between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present 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) hybridization L% Ficoll, 42.
  • denaturing agents such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42.
  • hybridization occurs only when the identity between the two sequences is at least 95%, and more preferably 97%, and the polypeptide encoded by the hybridizable polynucleotide is SEQ ID NO:
  • the mature polypeptide shown in 2 has the same biological function and activity.
  • 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 DNA mismatch repair gene protein 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 DNA mismatch repair gene protein 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) separating the double-stranded DNA sequence from the DM of the genome; 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 DNA sequences is often the method of choice. The more commonly used method is the separation of cDM sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library. There are many mature techniques for extracting mRNA, and kits are also commercially available (Qiagene).
  • 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 Clontech. When polymerase reaction technology is used in combination, even very small expression products can be cloned.
  • genes can be screened from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or loss of marker gene function; (3) determination Level of transcript of human DNA mismatch repair gene protein 13; (4) Detecting the protein product of gene expression by immunological techniques or measuring the biological activity of the foot. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is usually a DM sequence chemically synthesized based on the gene sequence information of the present invention.
  • the gene itself or the fragment of the present invention can of course be used as a probe: a DNA probe can be labeled with a radioisotope, luciferin, or an enzyme (such as alkaline phosphatase).
  • the protein product of human DNA mismatch repair gene protein 13 gene can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method using PCR technology to amplify DM / RNA is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-rapid amplification of cDNA ends
  • the primers used for PCR may be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • the polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467): Nucleotide 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 the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector of the present invention or directly using human DNA mismatch repair gene protein 13 coding sequence, and the recombinant technology to produce the polypeptide of the present invention Methods.
  • a polynucleotide sequence encoding a human DNA mismatch repair gene protein 13 may be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al.
  • any plasmid and vector can be used to construct recombinant expression vectors.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translation control elements.
  • DM sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis.
  • promoters are: the lac or trp promoter of E.
  • eukaryotic promoters include the CMV immediate early promoter, HSV thymidine kinase promoter, and early and late SV40 promoters Promoters, retroviral LTRs and other known promoters that control the expression of prokaryotic or eukaryotic cells or their viruses.
  • Expression vectors also include translation initiation, ribosome binding sites for f, and transcription terminators: Inserting enhancer sequences into the vector will enhance its transcription in high eukaryotic cells. Enhancers are cis-acting factors expressed by DM, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Examples include 100 to 270 base pair SV40 enhancers at the late side of the replication origin, polyoma enhancers and adenovirus enhancers at the late origin.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture. Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli, etc.
  • 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 etc.
  • a polynucleotide encoding human DM mismatch repair gene protein 13 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to form a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • host cell refers to a prokaryotic cell, such as a bacterial cell; or a eukaryotic cell, such as a salamander, 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
  • organic cells insect cells such as fly S2 or Sf9
  • animal cells such as CH0, COS, or Bowes melanoma Transformation of a host cell by the DNA sequence of the present invention or a recombinant vector containing the DNA sequence
  • the host is a prokaryote such as E. coli? 7.
  • Competent cells capable of absorbing DM can be harvested after exponential growth and treated with CaCl. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, the transformation can also be done by electricity. Law.
  • the host is a eukaryote
  • the following DNA transfection methods can be used: calcium phosphate coprecipitation: or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human DNA mismatch repair gene protein 13 (Science, 1984; 224: 1431). Generally, the following steps are followed:
  • polynucleotide (or variant) encoding human human DNA mismatch repair gene protein 13 of the present invention or transforming or transducing a suitable host cell with a recombinant expression vector containing the polynucleotide:
  • 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, 7-cell dialysis bacteria, 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, 7-cell dialysis bacteria, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, Ion exchange chromatography, high performance liquid chromatography
  • FIG. 1 is a comparison diagram of gene chip expression profiles of human DNA mismatch repair gene protein 13 and human DNA mismatch repair gene protein MSH3 of the present invention.
  • the upper graph is a graph of the expression profile of human DNA mismatch repair gene protein 13, and the lower graph is the graph of the expression profile of human DNA mismatch repair gene protein MSH3.
  • 1 indicates fetal kidney
  • 2 indicates fetal large intestine
  • 3 indicates fetal small intestine
  • 4 indicates fetal muscle
  • 5 indicates fetal brain
  • 6 indicates fetal bladder
  • 7 indicates unstarved L02
  • 8 indicates L02 +, lhr, As 3+
  • 9 indicates ECV304 PMA-
  • 10 means ECV304 PMA +
  • 11 means fetal liver
  • 12 means normal liver
  • 13 means thyroid
  • 14 means skin
  • 15 means fetal lung
  • 16 means lung
  • 17 means lung cancer
  • 18 means fetal spleen
  • 19 means spleen
  • 20 Indicates prostate
  • 21 indicates fetal heart
  • 22 indicates heart
  • 23 indicates muscle
  • 24 indicates testis
  • 25 indicates fetal thymus
  • 26 indicates thymus.
  • Figure 2 is a polyacrylamide gel electrophoresis image of isolated human DNA mismatch repair gene protein 13 (SDS- PAGE), 13KDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
  • CDNA was synthesized using fetal brain cell total RNA as a template and oiigo-dT as a primer for reverse transcription reaction. After purification by Qiagene's kit, the following primers were used for PCR amplification:
  • Primerl 5'- GCTGTGCCTGGCCACAATGTTACC-3, (SEQ ID NO: 3)
  • Primer2 5'- TTCTTGTAACACCGTTAGGCGTTT -3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp at the 5 ′ end of SEQ ID NO: 1;
  • Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
  • Amplification reaction conditions 50 leg ol / L KC1, 10ramol / L Tris- in a reaction volume of 50 ⁇ 1
  • RNA RNA was synthesized by electrophoresis on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-ImM EDTA- 2.2M formaldehyde, and then transfer to On nitrocellulose membrane.
  • the DNA probe used was the sequence of the 13 coding region (1366b P to 1725bp) of the human DNA mismatch repair gene protein amplified by PCR as shown in FIG. 1.
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH, P0 4 (pH 7.4)-5 x SSC- 5 x Denhardt's solution and 200 ⁇ g / mi salmon sperm DNA. After hybridization, the filters were 55 in 1 ⁇ SSC-0.1% SDS. C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant human DNA mismatch repair gene protein 13
  • Primer 3 5,-CCCCATATGATGTTCCCCTCATCCAGACGCTGG -3 '(Seq ID No: 5)
  • Primer4 5'- CCCGAATTCTCACAGGATCCTTTGGGTGTCTCT -3, (Seq ID No: 6)
  • the 5' ends of these two primers contain Ndel and EcoRI digestion sites, respectively , followeded by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the Ndel and EcoRI restriction sites correspond to the selectivity on the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Endonuclease site.
  • the pBS-0479d01 plasmid containing the full-length target gene was used as a template for the PCR reaction.
  • the PCR reaction conditions are as follows: a total volume of 50 ⁇ 1 contains 10 pg of pBS-0479d01 plasmid, bow! Thing! ⁇ ! ⁇ And? ! ⁇ ! ⁇ :! :-Separate it! ! for! ⁇ ⁇ , Advantage polymerase Mix (Clontech) 1 ⁇ 1. Cycle parameters: 94 ° C 20s, 60 ° C 30s, 68 ° C 2 min, a total of 25 cycles.
  • Ndel and EcoRI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase.
  • the ligated product was transformed with colibacillus DH50 by the calcium chloride method, containing kanamycin (Final concentration 30 g / ml) LB plate was cultured overnight, and K-type clones were selected by colony PCR method. ⁇ Sequencing was performed to select positive clones with correct sequence (pET-0479d01).
  • the recombinant plasmid was transformed into E. coli BL2UDE3 by the calcium chloride method ) plySs (Novagen).
  • NH2-Met-Phe-Pro-Ser-Ser-Arg-Arg-Trp-Cys-Leu-Gln-Trp-Lys-Pro-Pro-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. Immunochemistry, 1969; 6:43. Rabbits were immunized with 4 mg of the above-mentioned cyanoprotein multi-month complex plus complete Freund's adjuvant, and 15 days later the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost the immunity once.
  • a titer plate coated with a 15 g / ml bovine serum albumin peptide complex was used for ELISA to determine the titer of antibody in serum.
  • Protein A-Sepharose was used to isolate total IgG from antibody-positive home-immunized serum.
  • the peptide was bound to a cyanogen bromide-activated Sepharose 4B column, and the peptide and antibody were separated from the total IgG by affinity chromatography. Immunoprecipitation proves that the purified antibody can specifically bind to human DNA mismatch repair protein 13.
  • Example 6 Application of the polynucleotide fragment of the present invention as a hybridization probe
  • Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways.
  • the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to Identify whether it contains the polynucleotide sequence of the present invention and detect a homologous polynucleotide sequence, further use the probe to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissues, 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.
  • Filter hybridization methods include dot blotting, Southern 3 ⁇ 4 Trace method, Nor thern blot method, and copy method, etc., all are used to fix the polynucleotide sample to be tested on the filter and then use basically the same step hybridization.
  • 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 synthesized 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 probe is 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 same or complementary oligonucleotide fragment of the polynucleotide SEQ ID NO: 1 is used in this example.
  • the dot blot method is used to fix the sample on the filter membrane. Under high-intensity washing conditions, the first type of probe and the The sample has the strongest hybridization specificity and is retained.
  • oligonucleotide fragments for use as hybridization probes from the polynucleotide SEQ ID NO: 1 of the present invention 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 The regions are compared for homology. If the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used;
  • Probe 1 which belongs to the first type of probe, is completely inserted with the gene chip of SEQ ID NO: 1 Homologous or complementary (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 membranes nitrocellulose membranes
  • Gene chip or DNA microarray is a new technology that many national laboratories and major pharmaceutical companies are currently developing and developing. It refers to the orderly and high-order arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as a target DM for gene chip technology for high-throughput research of new gene functions; search for and screen tissues 4 heterogeneous new genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as heredity disease.
  • the specific method steps have been reported in the literature. For example, see DeRisi, LL., Lyer, V. & Brown, P.0. (1997) Science 278, 680-686. And Helle, RA, Schema, M. ., Chai, A., Shalom, D., (1997) PNAS 94: 2150-21
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs as target DNA, including the present invention Polynucleotide. They were amplified by PCR respectively. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium with a Cartesian 7500 spotting instrument (purchased from Cartesian, USA). The distance is 280 ⁇ . The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to fix the DNA on the glass slides to prepare chips.
  • the specific method steps have been reported in the literature in various ways. The post-sampling processing steps of this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) by a single method, and raRNA was purified with Oligotex mRNA Midi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP 5-Amino-propargy 2'-deoxyuridine 5'-triphate coupled to Cy3 fluorescent dye, purchased from Amersham Phamacia Biotech Company, labeled mRNA of human mixed tissues, using a fluorescent reagent Cy5dUTP (5- Amino- propargy 1-2'- deoxyur id ine 5'-tr iphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech company, labeled the specific tissue (or stimulated cell line) raRNA of the body, and purified the probe to prepare the probe.
  • Cy3dUTP 5-Amino-propargy 2'-deoxyuridine 5'-triphate coupled to Cy3 fluorescent dye, purchased from Amersham Phamaci
  • Probes from the two types of tissues and chips were hybridized in a UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, washed with a washing solution (1> ⁇ SSC, 0.2% SDS) at room temperature, and then scanned with ScanArray.
  • the 3000 scanner purchased from General Scanning, USA was used for scanning. The scanned image was analyzed and processed with Imagene software (Biodiscovery, USA) to calculate the Cy3 / Cy5 ratio of each point.
  • the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA-Ecv304 cell line, and non-starved L02 Cell line, arsenic stimulated L02 cell line for 1 hour, arsenic stimulated L02 cell line for 6 hours prostate, heart, lung cancer, fetal bladder, fetal small intestine, fetal large intestine, fetal thymus, fetal muscle, fetal liver, fetal kidney, fetal spleen, Fetal brain and fetal lung with 3 ⁇ 4 fetal heart.
  • polypeptides of the present invention can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection and immune-free diseases, etc. .
  • the repair process consists of a variety of mut genes (such as MutH, MutL, Mu t S and Mu t U) and various single-stranded DNA binding proteins.
  • the repair system is involved in the repair of DNA mismatch bases.
  • the DNA mismatch repair gene Mu t S forms a family of proteins.
  • the human DNA mismatch repair genes Mut S such as hMSH2 gene and MSH 3 gene, are closely related to the occurrence of hereditary colon polyp tumors, blood system-related malignancies such as leukemia, and hereditary diseases.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of the human DNA mismatch repair gene protein MSH 3-both have similar biological functions. It is mainly involved in DNA mismatch repair in the body and is important for the correct expression of genetic information. Its abnormal expression is usually closely related to the development of embryonic disorders, cell division and proliferation, immune system abnormalities, and related diseases.
  • the abnormal expression of the human DNA mismatch repair gene protein 1 3 of the present invention will produce various diseases, especially various tumors, embryonic developmental disorders, growth and development disorders, inflammation, immune-related diseases, these Illnesses include, but are not limited to:
  • Tumors of various tissues gastric cancer, liver cancer, lung cancer, esophageal cancer, breast cancer, leukemia, lymphoma, thyroid tumor, uterine fibroids, neuroblastoma, astrocytoma, ependymoma, glioblastoma, nerve Fibroma, colon cancer, melanoma, bladder cancer, uterine cancer, endometrial cancer, colon cancer, thymic tumor, nasopharyngeal cancer, laryngeal cancer, tracheoma, fibroma, fibrosarcoma, lipoma, liposarcoma
  • Fetal developmental disorders congenital abortion, cleft palate, limb loss, limb differentiation disorder, atrial septal defect, neural tube defect, congenital hydrocephalus, congenital glaucoma or cataract, congenital deafness
  • Growth and development disorders mental retardation, brain development disorders, skin, fat, and muscular dysplasia, bone and joint dysplasia, various metabolic defects, stunting, dwarfism, Cushing's syndrome
  • Sexual retardation Inflammation chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations
  • Immune diseases Systemic lupus erythematosus, rheumatoid arthritis, bronchial asthma, urticaria, specific dermatitis, post-infection myocarditis, scleroderma, myasthenia gravis, Guillain-Barre syndrome, common variable immunodeficiency disease , Primary B-lymphocyte immunodeficiency disease, Acquired immunodeficiency syndrome
  • the abnormal expression of the human DNA mismatch repair gene protein 1 3 of the present invention will also produce certain hereditary, hematological diseases and the like.
  • the polypeptide of the present invention can be directly used in the treatment of diseases-for example, it can treat various diseases, especially various tumors, embryonic development disorders, growth disorders, inflammation, immunity Sexual diseases, certain hereditary, blood diseases, etc.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human DNA mismatch repair gene protein 1 3.
  • Agonists improve human DNA mismatch repair gene protein 1 3 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing human DNA mismatch repair gene protein 1 3 can be cultured together with labeled human DNA mismatch repair gene protein 1 3 in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human DNA mismatch repair gene protein 13 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human DNA mismatch repair gene protein 13 can bind to human DNA mismatch repair gene protein 13 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide to make the Peptides cannot perform biological functions.
  • human DNA mismatch repair gene protein 13 can be added to the bioanalytical assay, and the determination of the compound's interaction with human DM mismatch repair gene protein 13 and its receptor I 'Division Influence to determine if a compound is an antagonist.
  • Receptor deletions and analogs that act as antagonists can be screened in the same manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to human DNA mismatch repair gene protein 1 3 can be obtained by screening a library of organic peptides composed of various possible combinations of amino acids bound to a solid phase. During screening, the human DNA mismatch repair gene protein 13 molecules should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, fragments, derivatives, analogs or their cells as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the human DM mismatch repair gene protein 1 3 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fa b fragments and Fa b Library-generated snippets.
  • polyclonal antibodies can be obtained by directly immunizing animals (such as home immunity, mice, rats, etc.) with human DNA mismatch repair gene protein 13.
  • Various adjuvants can be used to enhance the immune response, including but not limited to 'S adjuvant and so on.
  • Techniques for preparing monoclonal antibodies to human DNA mismatch repair gene protein 13 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that combine human constant regions with non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851).
  • Existing techniques for producing single-chain antibodies (US Pat No .4946778) can also be used to produce single-chain antibodies against human DNA mismatch repair gene protein 13.
  • Antibodies against human DNA mismatch repair gene protein 13 can be used in immunohistochemical techniques to detect human DNA mismatch repair gene protein 13 in biopsy specimens.
  • Monoclonal antibodies that bind to human DNA mismatch repair gene protein 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 DNA mismatch repair gene protein 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 DNA mismatch repair gene protein 13 positive Cell.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human DNA mismatch repair protein E protein 13.
  • Administration of an appropriate dose of antibody can stimulate or block the production or activity of human DM mismatch repair gene protein 13.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human DNA mismatch repair gene protein 13 levels.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human DNA mismatch repair gene protein 13 detected in the test can be used to explain the importance of human DNA mismatch repair gene protein 13 in various diseases and to diagnose the role of human DNA mismatch repair gene protein 13 disease.
  • 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 DM mismatch repair gene protein 13 can also be used for a variety of therapeutic purposes.
  • Gene therapy technology can be used to treat non-expression or abnormality / inactivity due to human DNA mismatch repair gene protein 13 Abnormal cell proliferation, development, or metabolism caused by expression.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human DNA mismatch repair gene protein 1 3 to inhibit endogenous human DNA mismatch repair gene protein 1 3 activity.
  • a mutated human DNA mismatch repair gene protein 1 3 may be shortened, and the human DM mismatch repair gene protein 1 3 is missing the signaling functional domain. Although it can bind to downstream substrates, it lacks signal transduction. active.
  • the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human DNA mismatch repair gene protein 13.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc. can be used to transfer a polynucleotide encoding human DNA mismatch repair gene protein 1 3 into a cell.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human I) NA mismatch repair gene protein 13 can be found in existing literature (Sambrook, etal.).
  • a recombinant polynucleotide encoding human DNA mismatch repair gene protein 1 3 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit human DNA mismatch repair gene protein 1 3 raRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphoramidite chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA.
  • This DNA sequence has been integrated downstream of the vector's RNA polymerase promoter.
  • it can be decorated in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human DM mismatch repair gene protein 1 3 can be used for the diagnosis of diseases related to human DNA mismatch repair gene protein 1 3.
  • the polynucleotide encoding human DNA mismatch repair gene protein 1 3 can be used to detect the expression of human DNA mismatch repair gene protein 1 3 or the abnormal expression of human DNA mismatch repair gene protein 1 3 in a disease state.
  • the DNA sequence encoding human DNA mismatch repair gene protein 1 3 can be used to hybridize biopsy specimens to determine the expression status of human DNA mismatch repair gene protein 1 3.
  • Hybridization techniques include Sout hern blotting, Nor thern blotting, in situ hybridization, and the like. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • polynucleotides of the present invention can be used as probes to be fixed on a microarray (Microcroix) or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis of genes and genes in tissues diagnosis. Repairing genes with human DNA mismatches Protein 13 specific primers can be amplified by RNA-polymerase chain reaction (RT-PCR) in vitro to detect the transcription product of human DNA mismatch repair gene protein 13.
  • RT-PCR RNA-polymerase chain reaction
  • Detection of mutations in the human DNA mismatch repair gene protein 13 gene can also be used to diagnose human DNA mismatch repair gene protein 13-related diseases.
  • Human DM mismatch repair gene protein 13 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human DNA mismatch repair gene protein 13 DNA sequence. Mutations can be detected using existing techniques such as Southern blotting, DNA sequencing, 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 marking chromosome positions.
  • the important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared based on cDNA, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those hybrid cells containing human genes corresponding to the primers produced amplified fragments.
  • the somatic hybrid cell PCR mapping method is a fast method for locating DNA to a specific chromosome.
  • oligonucleotide primers of the present invention by a similar method, a group of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sub- Positioning.
  • 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 a single 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, Mendeian Inheritance in Man (available online with Johns Hopkins University Welch Medical Library). Linkage analysis can then be used to determine the relationship between genes and diseases that have been mapped to chromosomal regions.
  • the differences in cDNA or genomic sequences between the affected and unaffected individuals need to be determined. If a mutation is observed in some or all diseased individuals and the mutation is not observed in any normal individuals, the mutation may be the cause of the disease. Comparing sick and coming sick individuals usually involves looking for staining first Structural changes in the body, such as deletions or translocations that are visible from the chromosomal level or detectable with cDNA sequence-based PCR. According to the current physical mapping and the resolution capabilities of the basic SJ mapping technology, the cDNA that is accurately positioned to the disease-related chromosomal region can be one of 50 to 500 potentially pathogenic genes (assuming 1 trillion bases) Base mapping ability and each 20k b 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 containing one or more ingredients of the pharmaceutical composition of the present invention.
  • the polypeptide of the present invention can be used in combination with a therapeutic compound thereof.
  • 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 DNA mismatch repair gene protein 1 3 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human DNA mismatch repair gene protein 1 3 to be administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician.

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Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine de réparation 13 du mésappariement de l'ADN, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, des troubles du développement, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la protéine humaine de réparation 13 du mésappariement de l'ADN.
PCT/CN2001/000369 2000-03-22 2001-03-19 Nouveau polypeptide, proteine humaine de reparation 13 du mesappariement de l'adn, et polynucleotide codant pour ce polypeptide Ceased WO2001070960A1 (fr)

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CN 00115023 CN1314382A (zh) 2000-03-22 2000-03-22 一种新的多肽——人dna错配修复基因蛋白13和编码这种多肽的多核苷酸
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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [online] 2 March 2000 (2000-03-02), Database accession no. AL133247 *
DATABASE GENBANK [online] 21 December 1999 (1999-12-21), Database accession no. AC004941 *
DATABASE GENBANK [online] 21 December 1999 (1999-12-21), Database accession no. AC005008 *
DATABASE GENBANK [online] 21 December 1999 (1999-12-21), Database accession no. AC006153 *
DATABASE GENBANK [online] 23 February 2000 (2000-02-23), Database accession no. AC016138 *

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