WO2001088146A1 - Nouveau polypeptide, alkylguanine-adn/cysteine proteine methyltransferase humaine 11, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, alkylguanine-adn/cysteine proteine methyltransferase humaine 11, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001088146A1
WO2001088146A1 PCT/CN2001/000464 CN0100464W WO0188146A1 WO 2001088146 A1 WO2001088146 A1 WO 2001088146A1 CN 0100464 W CN0100464 W CN 0100464W WO 0188146 A1 WO0188146 A1 WO 0188146A1
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Prior art keywords
polypeptide
polynucleotide
human
dna
protein cysteine
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PCT/CN2001/000464
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English (en)
Chinese (zh)
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Yumin Mao
Yi Xie
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Shanghai Biowindow Gene Development Inc.
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Priority to AU62004/01A priority Critical patent/AU6200401A/en
Publication of WO2001088146A1 publication Critical patent/WO2001088146A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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 alkylated-DM-protein cysteine methyltransferase 11, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide.
  • DNA molecules require a high degree of accuracy and integrity. No molecule in a cell can be compared to it.
  • organisms have not only evolved systems that can correct accidental replication errors, but also exist systems that can repair damage to DNA molecules caused by environmental factors (such as rays) and chemicals in the body, such as photoresurrection repair systems, Excision repair system, recombination repair system, SOS repair system, repair of basalization damage.
  • photoresurrection repair systems In order to deal with the invasion of foreign DNA, some organisms have also evolved restriction modification systems. To date, almost every organism that has been tested has extremely effective DNA repair mechanisms to ensure that specific alkylated oxygen does not accumulate in the genome.
  • This phosphonium-based damage repair pathway makes cells more resistant to the harmful effects of chemical mutagens and radiation. Hydrants covalently modify the genome of cells to produce miscoded base derivatives and damage that hinder DNA replication. Its main mutagenesis and carcinogenic effect on DM is the formation of 0-6-alkylguanine. For 06-- alkyl with guanine is restored by 06-- methylguanine - DNA methyl transferases (alkylated -DM - protein-cysteine methyltransferase) (of MGMT) is completed. The removed methyl groups bind to two cysteine residues of the enzyme. However, enzymes that have been methylated cannot be converted to unmethylated enzymes. This phenomenon is called suicide behavior of the enzyme. Thus, each removal of a methyl group from a DNA molecule consumes a new enzyme molecule. Most, but not all, MGMTs are also capable of repairing 0 4 -methylthymine.
  • N-terminal and C-terminal regions of MGMT in mammals are 50% conserved, and one of the regions containing 68 amino acid residues is extremely conserved, with a conservation rate of up to 90%, which contains a hexapeptide fragment [LIVMF] -PCHR- [LIVMF] is used as a characteristic sequence template.
  • This fragment is present in MGMT of various known organisms, and its cysteine residue is the active site of the methyl receptor.
  • Human MGMT is an important DNA repair enzyme with a molecular weight of 24KD. It can transfer 0 6 -guanine and 0 4 -alkylthymine residues in DNA to cysteine residues at the active site of the enzyme. Base (cysteine 167).
  • the alkylated nucleotide residues are composed of carcinogenic methylated agents (N-methylnitrosuria (should U), N-nitroso compounds (NEU), and N, N-dialkyl Nitrosamines), these alkylated nucleotide residues will Mutations in the base sequence of the DNA sequence due to its incorrect coding.
  • Many human genes involved in tumor formation cause cancer due to point mutations, such as the ras gene, the p53 gene, and the ERCC-3 gene.
  • the mutations that occur are thought to be critical for the activation (oncogenes) or suppression (oncogenes) of these genes, and there is ample evidence that point mutations are caused by the action of alkylated agents on MA in specific human cancer etiology .
  • MGMT can specifically repair DNA damage caused by sulfonating agents, the level of this enzyme in cells is considered to be a key factor determining the sensitivity of cells to the tumor-induced mutation effect of alkylating carcinogens
  • Polypeptides containing the above-mentioned conservative hexapeptides and their antagonists, agonists and inhibitors can be used for tumor diagnosis, prediction and treatment of cancers caused by alkylated carcinogens. For example, it treats patients with telangiectasias who have cancer due to mutations in the gene P53 and patients with Li-Fraumeni who have a tendency to develop cancer.
  • the human alkylated-DNA-protein cysteine methyltransferase 11 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.
  • Newcomers The isolation of the alkylated-DNA-protein cysteine methyltransferase 11 protein encoding gene also provides a basis for research to determine the role of this protein in health and disease states. This protein may form the basis for the development of diagnostic and / or therapeutic drugs for the disease, so it is important to isolate its coding DNA.
  • 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 alkylated-DM-protein cysteine phosphotransferase 11.
  • Another object of the present invention is to provide a cysteine methyltransferase containing a human alkylated-DM-protein. Genetically engineered host cell for a polynucleotide transferase 11.
  • Another object of the present invention is to provide a method for producing human alkylated-DM-protein cysteine methyltransferase 11.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention, human alkylated-DM-protein cysteine methyltransferase 11.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors directed to the polypeptide of the present invention, human fluorenated-DNA-protein cysteine methyltransferase 11.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases related to abnormalities in human alkylated-DNA-protein cysteine phosphotransferase 11. Summary of invention
  • 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 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 1597-1911 in SEQ ID NO: 1; and (b) a sequence having 1-1925 in SEQ ID NO: 1 Sequence of bits.
  • 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 alkylated-DNA-protein cysteine methyltransferase 11 protein, 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 alkylated-DM-protein cysteine methyltransferase 11 protein in vitro, which comprises detecting the polypeptide or its encoding in a biological sample. A mutation in a polynucleotide sequence, or the amount or biological activity of a polypeptide of the invention in a biological sample is detected.
  • the present invention also relates to a pharmaceutical composition, which contains the polypeptide of the present invention or a mimic, activator, antagonist Antibiotics or inhibitors and pharmaceutically acceptable carriers.
  • the present invention also relates to the polypeptides and / or polynucleotides of the present invention prepared for the treatment of cancer, developmental or immune diseases or other abnormal expression due to human alkylated-DNA-protein cysteine methyltransferase 11 Use of medicines that cause disease.
  • FIG. 1 is a comparison diagram of gene chip expression profiles of the alkylated-DNA-protein cysteine methyltransferase 11 and human fluorinated-DNA-protein cysteine methyltransferase 12 of the present invention.
  • the upper graph is a graph of the expression profile of human adenylated-DNA-protein cysteine methyltransferase 11 and the lower graph is the expression profile of human alkylated-DNA-protein cysteine methyltransferase 12 Histogram.
  • 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 non-starved 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 is the prostate
  • 21 is the fetal heart
  • 22 is the heart
  • 23 is the muscle
  • 24 is the testis
  • 25 is the fetal thymus
  • 26 is the thymus.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of the isolated human alkylated-DM-protein cysteine glutamyl transferase 11.
  • l lkDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a protein or polynucleotide “variant” refers to a protein or polynucleotide that has one or more amino acid or nucleotide changes Amino acid sequence or 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 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 means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with human alkylated-DM-protein cysteine methyltransferase 11, 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 alkylated-DNA-protein cysteine methyltransferase 11.
  • Antagonist refers to a type that blocks or regulates human alkylated-DM-protein cysteamine when combined with human alkylated-DM-protein cysteine methyltransferase 11.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind to human adenylated-DNA-protein cysteine methyltransferase 11.
  • Regular refers to a change in the function of human alkylated-DM-protein cysteine glutamyl transferase 11, including an increase or decrease in protein activity, a change in binding characteristics, and a human alkylated-DNA-protein half Changes in any other biological, functional or immune properties of cystine methyltransferase 11.
  • Substantially pure ' means essentially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated.
  • Those skilled in the art can purify human alkylated-DNA-protein half using standard protein purification techniques. Cystine methyltransferase 11. Basically pure human alkylated-DM-protein cysteine methyltransferase 11 produces a single main band on a non-reducing polyacrylamide gel. Human alkyl The purity of the N-DNA-protein cysteine methyltransferase 11 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to polynucleotides that naturally bind through base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence "C-TG-A” can be combined with the complementary sequence "G-ACT”.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands The efficiency and strength of hybridization between nucleic acid strands has a significant effect.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Southern imprinting or Nor thern blotting, etc.) under conditions of reduced stringency.
  • Substantially homologous sequences or hybridization probes can compete and inhibit the binding of fully homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences bind to each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are the same or similar in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, such as through the MEGALIGN program
  • the MEGALIGN program can compare two or more sequences (Hi gg ins, D. G. and
  • the percent identity between nucleic acid sequences can also be determined by the Clus ter method or by methods known in the art such as Jotun Hein (Hein J., (1990) Methods in enzymology 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 substitution for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DM or RM sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to HFP or a chemical modification of its nucleic acid. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa, F (ab ') 2 and Fv, which can specifically bind to the antigenic determination of human alkylated-DNA-protein cysteine methyltransferase 11 cluster.
  • Humanized antibody means that the amino acid sequence of a non-antigen binding region is replaced with a human antibody Antibodies that are more similar but still retain their 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 alkylated-DM-protein cysteine methyltransferase 11 means that human alkylated-DM-protein cysteine methyltransferase 11 is substantially free of natural and Related to other proteins, lipids, sugars or other substances. Those skilled in the art will be able to purify human amylated-DM-protein cysteine methyltransferase 11 using standard protein purification techniques. Substantially pure polypeptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of the human alkylated-DM-protein cysteine methyltransferase 11 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide ⁇ ⁇ ⁇ -DM-protein cysteine methyltransferase 11, 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 polypeptide of the present invention may be a naturally purified product, or a chemically synthesized product, or produced from a prokaryotic or eukaryotic host (eg, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology.
  • polypeptides of the invention may be glycosylated, or they may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the initial methionine residue.
  • the invention also includes fragments, derivatives and analogs of human alkylated-DNA-protein cysteine methyltransferase 11.
  • fragment refers to substantially maintaining the same biological function of the human alkylated-DNA-protein cysteine methyltransferase 11 of the present invention or Active peptide.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or ( ⁇ ) a type in which a group on one or more amino acid residues is replaced by another group to include a substituent; or ( ⁇ ⁇ )
  • the mature polypeptide and another compound such as elongation Fusion of a polypeptide half-life compound, such as polyethylene glycol; or (IV) a polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence used to purify the polypeptide) in which an additional amino acid sequence is fused into a mature polypeptide Or proteinogen sequence).
  • such fragments, derivatives and analogs are considered to be within the knowledge of those skilled in the art.
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes the nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a CDM library of human fetal brain tissue. It contains a polynucleotide sequence of 1925 bases in length and its open reading frame 1597-1911 encodes 104 amino acids.
  • this polypeptide has a similar expression profile to human alkylated-DNA-protein cysteine methyltransferase 12, and it can be deduced that the human alkylated-DM-protein cysteine Methyltransferase 11 has a similar function to human alkylated-DM-protein cysteine methyltransferase 12.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • a "degenerate variant" refers to a nucleic acid sequence encoding a protein or polypeptide having SEQ ID NO: 2 but different from the coding region sequence shown in SEQ ID NO: 1 in the present invention.
  • the polynucleotide encoding the mature polypeptide of SEQ ID NO: 2 includes: only the coding sequence of the mature polypeptide; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences); Coding sequence.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to variants of the polynucleotides described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • Variants of this polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the polypeptide it encodes .
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences).
  • the 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) Cross When using a denaturant, such as 50. /. (V / v) formamide, 0.1% calf serum / 0.1 l F icol l, 42.
  • 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 nuclei. 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 adenylated-DNA-protein cysteine methyltransferase 11.
  • 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 alkylated-DM-protein cysteine methyltransferase 11 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 DM of the genome; 2) chemically synthesizing the DM sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DM sequences is often the method of choice.
  • the more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating 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.
  • Various methods have been used to extract mRNA, and kits are also commercially available (Qiagene). It is also a common method to construct a CDM library (Sambrook, et al., Molecular Cloning, A Laboratory Manua, Cold Spruing Harbor Laboratory. New York, 1989).
  • Commercially available cDM 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 of the present invention can be screened from these cDM 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 alkylation-DM-protein cysteine methylation Level of transcript of transferase 11; (4) Detecting protein products of gene expression by immunological techniques or measuring biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 Nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used 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.
  • DM probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of the human alkylated-DM-protein cysteine methyltransferase 11 gene can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunoassay. Adsorption method (ELISA) and so on.
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunoassay.
  • ELISA Adsorption method
  • a method for amplifying DM / RNA by PCR is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-Rapid Amplification of cDNA Ends
  • the primers used for PCR can be appropriately based on the polynucleotide sequence information of the present invention disclosed herein Select and synthesize using conventional methods.
  • the amplified DNA / RM fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DM fragments and the like obtained as described above can be measured by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, sequencing needs to be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell that is genetically engineered using the vector of the present invention or directly using human amidated-DNA-protein cysteine adenyl transferase 11 coding sequence, and Method for producing a polypeptide of the present invention by recombinant technology.
  • a polynucleotide sequence encoding human alkylated-DM-protein cysteine methyltransferase 11 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 (Ros enberg, 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 translational regulatory elements.
  • Methods known to those skilled in the art can be used to construct expression vectors containing a DM sequence encoding human alkylated-DM-protein cysteine methyltransferase 11 and appropriate transcription / translation regulatory elements. These ones Methods include in vitro recombinant DM technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manua l, Cold Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on promoters to enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenoviral enhancers. '
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human alkylated-DNA-protein cysteine methyltransferase 11 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute the polynucleotide.
  • a host cell refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S2 or Sf9 animal cells
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DM can be harvested after the exponential growth phase and treated with the CaCl 2 method. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • the polynucleotide sequence of the present invention can be used to express or produce recombinant human amidated-DNA-protein cysteine methyltransferase 11 (Sc ience, 1984; 224: 1431). Generally there are the following steps:
  • polynucleotide or variant
  • encoding human human alkylated-DNA-protein cysteine methyltransferase 11 of the present invention, or a recombinant expression vector containing the polynucleotide for transformation or transformation Direct suitable host cells or a recombinant expression vector containing the polynucleotide for transformation or transformation Direct suitable host cells
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion Exchange chromatography, high performance liquid
  • polypeptides of the present invention as well as antagonists, agonists and inhibitors of the polypeptides, can be directly used in the treatment of diseases, for example, they can treat malignant tumors, adrenal deficiency, skin diseases, various types of inflammation, HIV infection, and immune diseases.
  • the methylated DM protein cysteine methyltransferase-specific conserved sequence is required to form its active mot if. It can be seen that the abnormal expression of the specific methylated DM protein cysteine methyltransferase mot if will cause the function of the polypeptide containing the mot if of the present invention to be abnormal, resulting in the repair of the alkylation damage of the DM molecule When abnormality occurs, the inheritance of genetic information transmission is generated, and related diseases such as tumors, embryonic developmental disorders, growth and development disorders, inflammation, etc. are generated.
  • the abnormal expression of the human alkylated-DM-protein cysteine methyltransferase 11 of the present invention will produce various diseases, especially various tumors, embryonic development disorders, growth disorders, and inflammation. These diseases 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 , Colon cancer, melanoma, adrenal cancer, bladder cancer, bone cancer, osteosarcoma, myeloma, bone marrow cancer, brain cancer, uterine cancer, endometrial cancer, cholangiocarcinoma, thymic tumor, nasal cavity and sinus cancer, nasopharyngeal cancer , Laryngeal cancer, tracheal tumor, fibroma, fibrosarcoma, lipoma, liposarcoma, leiomyoma
  • Embryonic disorders congenital abortion, cleft palate, limb loss, limb differentiation disorder, hyaline membrane disease, atelectasis, polycystic kidney, double ureter, cryptorchidism, congenital inguinal hernia, double uterus, vaginal atresia, suburethral Fissure, hermaphroditism, atrial septal defect, ventricular septal defect, pulmonary stenosis, arterial duct occlusion, neural tube defect, congenital hydrocephalus, iris defect, congenital cataract, congenital glaucoma or cataract, congenital deafness
  • Growth and development disorders mental retardation, cerebral palsy, brain development disorders, mental retardation, familial cerebral nucleus hypoplasia syndrome, strabismus, skin, fat and muscular dysplasia such as congenital skin sagging, premature Disease, congenital keratosis, various metabolic defects such as various amino acid metabolic defects, stunting, dwarfism, sexual retardation
  • Inflammation allergic reaction, bronchial asthma, allergic pneumonia, adult respiratory distress syndrome, sarcoidosis, rheumatoid arthritis, rheumatoid arthritis, osteoarthritis, cholecystitis, glomerulonephritis, immune complex Types of glomerulonephritis, acute anterior uveitis, dermatomyositis, urticaria, atopic dermatitis, hemochromatosis, polymyositis, Addison's disease, chronic active hepatitis, emergency bowel syndrome, atrophy Gastritis, systemic lupus erythematosus, myasthenia gravis, cerebrospinal multiple sclerosis, Guillain-Barre syndrome, intracranial granuloma, pancreatitis, myocarditis
  • the abnormal expression of the human adenylated-DM-protein cysteine methyltransferase 11 of the present invention will also cause certain heredit
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human alkylated-DM-protein cysteine methyltransferase 11.
  • Agonists increase human alkylated-DNA-protein cysteine methyltransferase 11 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing human amidated-DNA-protein cysteine methyltransferase 11 and a labeled human amidated-DNA-protein cysteine can be used in the presence of a drug. Methyltransferase 11 was cultured together. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human alkylated-DM-protein cysteine methyltransferase 11 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonists of human alkylated-DM-protein cysteine methyltransferase 11 can bind to human methylated-DM-protein cysteine methyltransferase 11 and eliminate its function, or inhibit the polypeptide Production, or binding to the active site of the polypeptide prevents the polypeptide from performing biological functions.
  • human alkylated-DNA-protein cysteine methyltransferase 11 can be added to the bioanalytical assay, and human alkylated-DNA-protein cysteine can be determined by measuring the compound The effect of the interaction between methyltransferase 11 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 manner as described above for screening compounds.
  • Polypeptide molecules capable of binding to human alkylated-DNA-protein cysteine methyltransferase 11 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 11-DNA-protein-cysteine methyltransferase 11 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 present invention also provides antibodies directed against human amidated-DNA-protein cysteine methyltransferase 11 epitopes. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • polyclonal antibodies can be obtained by direct injection of human amylase-DNA-protein cysteine methyltransferase 11 into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • adjuvants can be used to enhance Immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies to human alkylated-DM-protein cysteine methyltransferase 11 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.
  • Chimeric antibodies that bind human constant regions and non-human variable regions can be produced using existing techniques (Morr i son et al, PNAS, 1985, 81: 6851). And the existing technology for the production of single chain antibodies (US Pat No. 4946778) can also be used to produce anti-human amylase-DNA-protein cysteine alpha Single-chain antibody to glycosyltransferase 11.
  • Antibodies against human amylase-DM-protein cysteine methyltransferase 11 can be used in immunohistochemistry to detect human amylase-DM-protein cysteine methyltransferase 11 in biopsy specimens .
  • Monoclonal antibodies that bind to human alkylated-DNA-protein cysteine methyltransferase 11 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 alkylated-DNA-protein cysteine methyltransferase 11 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 amidated-DM-protein half Cystine methyltransferase 11 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human alkylated-DM-protein cysteine methyltransferase 11.
  • Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human alkylated-DNA-protein cysteine methyltransferase 11.
  • the invention also relates to a diagnostic test method for quantitative and localized detection of human alkylated-DNA-protein cysteine methyltransferase 11 levels.
  • tests are well known in the art and include FISH assays and radioimmunoassays.
  • the level of human alkylated-DM-protein cysteine methyltransferase 11 detected in the test can be used to explain human alkylated-DM-protein cysteine methyltransferase 11 in various diseases The importance and usefulness in diagnosing diseases in which human adenylated-DM-protein cysteine methyltransferase 11 functions.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • Polynucleotides encoding human alkylated-DNA-protein cysteine methyltransferase 11 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 adenylated-DM-protein cysteine methyltransferase 11. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human alkylated-DNA-protein cysteine methyltransferase 11 to inhibit endogenous human alkylated-DNA-protein half Cystine methyltransferase 11 activity.
  • 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 adenylated-DM-protein cysteine methyltransferase 11.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human alkylated-DNA-protein cyst
  • a variant human alkylated-DNA-protein cysteine methyltransferase 11 may be a shortened human alkylated-DM-protein cysteine methyltransferase lacking a signaling domain Enzyme 11, although it can bind to downstream substrates, lacks signal transduction activity. Therefore, recombinant gene therapy vectors are available For the treatment of diseases caused by abnormal expression or activity of human adenylated-DM-protein cysteine methyltransferase 11.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus, etc.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human alkylated-DM-protein cysteine methyltransferase 11 can be found in the existing literature (Sambrook, et al.).
  • Another polynucleotide encoding human alkylated-DNA-protein cysteine methyltransferase 11 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 phospho- DM-protein cysteine phosphotransferase 11 mRNA 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 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 RNA. This DNA 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 thiophosphate or peptide bonds instead of phosphodiester bonds.
  • the polynucleotide encoding human alkylated-MA-protein cysteine methyltransferase 11 can be used for diagnosis of diseases related to human alkylated-DNA-protein cysteine methyltransferase 11.
  • Polynucleotide encoding human alkylated-DNA-protein cysteine methyltransferase 11 can be used to detect the expression of human amidylated-DM-protein cysteine methyltransferase 11 or in a disease state Abnormal expression of human peptidyl-DM-protein cysteine methyltransferase 11.
  • the DNA sequence encoding human adenylated-DNA-protein cysteine methyltransferase 11 can be used to hybridize biopsy specimens to determine the expression of human alkylated-DNA-protein cysteine methyltransferase 11 situation.
  • Hybridization techniques include Southern 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.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DNA chip (also called a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human alkylated-DNA-protein cysteine can also be detected by in vitro amplification with human amidated-DNA-protein cysteine methyltransferase 11 specific primers for MA-polymerase chain reaction (RT-PCR) Transcript of amidinotransferase 11. Detection of mutations in the human peptidyl-DM-protein cysteine methyltransferase 11 gene can also be used to diagnose human alkylated-DM-protein cysteine pyltransferase 11-related diseases.
  • RT-PCR MA-polymerase chain reaction
  • Human alkylated-DNA-protein cysteine methyltransferase 11 mutations include point mutations compared to the normal wild-type human amylated-DM-protein cysteine methyltransferase 11 DNA sequence, Translocations, deletions, reorganizations, and any other abnormalities. Mutations can be detected using existing techniques such as Southern blotting, DM sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression, so Northern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will specifically target a specific position on a human chromosome and can hybridize to it. Currently, specific sites for each gene on the chromosome need to be identified. Currently, only a few chromosome markers based on actual sequence data (repeating polymorphisms) are available for marking chromosome positions. According to the present invention, in order to associate these sequences with disease-related genes, an important first step is to locate these DNA sequences on a chromosome.
  • PCR primers (preferably 15-35bp) are prepared according to cDM, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DM 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 CDM or genomic sequence differences 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 diseased and unaffected individuals usually involves first looking for structural changes in the chromosome, such as defects visible at the chromosomal level or detectable with cDM sequence-based PCR Missing or transposing. According to the resolution capabilities of current physical mapping and gene mapping technology, the cDNA accurately mapped to the chromosomal region associated with the disease can be one of 50 to 500 potentially pathogenic genes (assuming 1 megabase mapping resolution) Capacity and each 20kb corresponds to a gene).
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the invention.
  • these containers there may be instructional instructions given by government agencies that manufacture, use, or sell pharmaceuticals or biological products, which prompts permission for administration on the human body by government agencies that produce, use, or sell.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human adenylated-DM-protein cysteine methyltransferase 11 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human alkylated-DNA-protein cysteine methyltransferase 11 administered to a patient will depend on many factors, such as the mode of administration, the health conditions of the person to be treated, and the judgment of the diagnostician. Examples
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RM using Quik mRNA Isolat ion Kit (product of Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA.
  • a Smart cDNA cloning kit purchased from Clontech l ⁇ cDM fragment was inserted into the multicloning site of pBSK (+) vector (Clontech)) to transform DH5 a to form a cDNA library.
  • Dye terminate cycle react ion sequencing Kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Elmer
  • the determined cDNA sequences were compared with the existing public DNA sequence database (Genebank). When compared, one of the clones was found The CDM sequence of 0656f 05 is the new DM.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • CDNA was synthesized using fetal brain total RM as a template and ol igo-dT as a primer for reverse transcription reaction. After purification with Qiagene's kit, the following primers were used for PCR amplification:
  • Pr iraerl 5'- GTTTACAAATATCTGGTCGAATTG-3 '(SEQ ID NO: 3)
  • Primer2 5'- TTTTTTTTGTATTTTTAGTAGTGA-3 '(SEQ ID NO: 4)
  • Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
  • Primer2 is the 3, terminal reverse sequence of SEQ ID NO: 1.
  • a 50 ⁇ l reaction volume contains 50 mmo l / L KCl, 10 mmol / L Tris-HCl, pH 8. 5, 1. 5 mmol / L MgCl 2 , 200 mol / L dNTP, 1 Opmol primer, 1U of Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DM thermal cycler (Perkin-Elnier) for 25 cycles under the following conditions: 94. C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • ⁇ -act in was set as a positive control and template blank was set as a negative control.
  • the amplified product was purified using a QIAGEN kit and ligated to a P CR vector (Invitrogen) using a TA cloning kit.
  • DM sequence analysis results showed that the DM sequence of the PCR product was identical to the l-1925bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human amidated-DM-protein cysteine methyltransferase 11 gene expression
  • CAnal. Biochera 1987, 162, 156-159] 0 This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1), centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70 % ethanol, dried and dissolved in water.
  • a 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred at 42 ° C overnight in a solution containing 50% formamide-25mM KH 2 P0 4 (pH7. 4)-5 x SSC-5 x Denhardt's solution and 20 ( ⁇ g / ml salmon sperm DM. After hybridization, the filter was washed in 1 x SSC-0.1% SDS at 55 ° C for 30 minutes. Then Analysis and quantification using Phosphor Imager.
  • Example 4 In vitro expression, isolation and purification of recombinant human alkylated-DNA-protein cysteine methyltransferase 11
  • Primer 3 5'-CATGCTAGCATGACTGATCCTAGATGCTTATCA-3 '(Seq ID No: 5)
  • Pr imer4 5'-CATGGATCCTTAGTAGTGACGGGGTTTCACCAT-3' (Seq ID No: 6)
  • the 5 'ends of these two primers contain Nhel and BamHI digestion sites, respectively Points, followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the Nhel and BamHI restriction sites correspond to the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3) Selective endonuclease site.
  • the PCR reaction was performed using the pBS-0656f 05 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ 1 containing 10 pg of pBS-0656f05 plasmid, primers Primer-3 and Primer-4 were lOpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 4 ° 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 coliform bacteria DH5 cx by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 3 ( ⁇ g / ml)), positive colonies were screened by colony PCR method and sequenced. the positive clones with the correct sequence (PET- 0656f05) the recombinant plasmid by the calcium chloride method to transform E. coli BL21 (DE3) plySs (Nova g en products).
  • the host bacteria BL21 (pET-0656f 05) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 mmol / L, and the culture was continued for 5 hours.
  • the cells were collected by centrifugation, sonicated by ultrasound, and centrifuged The supernatant was collected and chromatographed using an affinity chromatography column His.
  • Bind Quick Cartridge product of Novagen capable of binding to 6 histidines (6His-Tag) to obtain purified human alkylated protein of interest- DNA-protein cysteine methyltransferase 11.
  • 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.
  • Protein A-Sepharose was used to isolate total IgG from antibody-positive rabbit serum.
  • the peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from the total IgG by affinity chromatography.
  • the immunoprecipitation method demonstrated that the purified antibody specifically binds to human alkylated-DM-protein cysteine methyltransferase 11.
  • 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.
  • Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They all use the same steps of hybridization after fixing the polynucleotide sample to be tested on the filter.
  • the sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer, so that the non-specific binding site of the sample on the filter is saturated with the carrier and the synthetic polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing the labeled probe and incubated to hybridize the probe to the target nucleic acid.
  • the unhybridized probes are removed by a series of membrane washing steps.
  • This embodiment utilizes higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Needle-to-sample hybridization has the strongest specificity and is retained.
  • oligonucleotide fragments from the polynucleotide SEQ ID NO: 1 of the present invention for use as hybridization probes should follow the following principles and several aspects to be considered:
  • the preferred range of probe size is 18-50 nucleotides
  • Those that meet the above conditions can be used as primary selection probes, and then further computer sequence analysis, including the primary selection probe and its source sequence region (ie, SEQ ID NO: 1) and other known genomic sequences and their complements For homology comparison of the regions, if the homology with the non-target molecular region is greater than 85% or there are more than 15 consecutive bases, the primary probe should not be used generally;
  • Probe 1 which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
  • Probe 2 which belongs to the second type of probe, is equivalent to the replacement mutation sequence (41Nt) of the gene fragment or its complementary fragment of SEQ ID NO: 1:
  • 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
  • the 32 P-Probe (the second peak is free ⁇ - 32 P-dATP) is prepared.
  • Gene chip or DNA microarray is a new technology that many national laboratories and large pharmaceutical companies are currently developing and developing. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of fast, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases .
  • the specific method steps have been reported in the literature. For example, refer to the literature DeRis i, JL, Lyer, V. & Brown, P. 0. (1997) Science 278, 680-686. And the literature Hel le, RA, Schema , M., Chai, A., Shalom, D., (1997) PNAS 94: 2150
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were respectively amplified by PCR, and the amplified product was purified to adjust its concentration to about 500ng / ul, and spotted on a glass medium with a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between them is 280 ⁇ m. The spotted slides were hydrated, dried, and cross-linked in a UV cross-linker. After elution, the slides were fixed to prepare DNA on a glass slide to prepare a chip. The specific method steps have been reported in the literature. The sample post-processing steps in this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and the mRNA was purified with Oligotex mRNA Midi Ki t (purchased from QiaGen).
  • Cy3dUTP (5-Amino-propargyl-2--deoxyuridine 5> -triphate cou led to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5- Amino-propargyl- 2 '-deoxyuridine 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 a probe.
  • Cy3dUTP (5-Amino-propargyl-2--deoxyuridine 5> -triphate cou led to Cy3
  • the probes from the above two tissues were hybridized with the chip in UniHyb TM Hybridization Solution (purchased from TeleChem) hybridization solution for 16 hours, and washed with a washing solution (lx SSC, 0.2% SDS) at room temperature. Scanning was then performed with a ScanArray 3000 scanner (purchased from General Scanning, USA). The scanned images were 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, non-starved L02 cell line, L02 cell line stimulated by arsenic for 1 hour, L02 cell line stimulated by arsenic 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, Fetal lung and fetal heart.

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Abstract

L'invention concerne un nouveau polypeptide, une alkylguanine-ADN/cystéine protéine méthyltransférase humaine 11, 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 l'alkylguanine-ADN/cystéine protéine méthyltransférase humaine 11.
PCT/CN2001/000464 2000-03-28 2001-03-26 Nouveau polypeptide, alkylguanine-adn/cysteine proteine methyltransferase humaine 11, et polynucleotide codant pour ce polypeptide WO2001088146A1 (fr)

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CN 00115226 CN1315536A (zh) 2000-03-28 2000-03-28 一种新的多肽——人烷基化－dna－蛋白半胱氨酸甲基转移酶11和编码这种多肽的多核苷酸

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Citations (1)

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US5879899A (en) * 1994-01-14 1999-03-09 National University Of Singapore Methods of assaying DNA-repairing enzymes and their alkylated derivatives

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5879899A (en) * 1994-01-14 1999-03-09 National University Of Singapore Methods of assaying DNA-repairing enzymes and their alkylated derivatives

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AU6200401A (en) 2001-11-26

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