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WO2001074995A2 - A novel polypeptide - human ataxia-telangiectasia mutant protein 15 and a polynucleotide encoding the same - Google Patents

A novel polypeptide - human ataxia-telangiectasia mutant protein 15 and a polynucleotide encoding the same Download PDF

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Publication number
WO2001074995A2
WO2001074995A2 PCT/CN2001/000230 CN0100230W WO0174995A2 WO 2001074995 A2 WO2001074995 A2 WO 2001074995A2 CN 0100230 W CN0100230 W CN 0100230W WO 0174995 A2 WO0174995 A2 WO 0174995A2
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WO
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Prior art keywords
polypeptide
polynucleotide
mutant protein
ataxia
sequence
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PCT/CN2001/000230
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French (fr)
Chinese (zh)
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WO2001074995A3 (en
Inventor
Yumin Mao
Yi Xie
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Biowindow Gene Development Inc. Shanghai
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Priority to AU89277/01A priority Critical patent/AU8927701A/en
Publication of WO2001074995A2 publication Critical patent/WO2001074995A2/en
Publication of WO2001074995A3 publication Critical patent/WO2001074995A3/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

Definitions

  • the present invention belongs to the field of biotechnology, and specifically, the present invention describes a new polypeptide, ataxia-capillary telangiectasia mutant protein 15, and a polynucleotide sequence encoding the polypeptide.
  • the invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
  • Ataxia-telangiectasia is a recessive disorder of frequently chromosomal body. Symptoms are ataxia of the cerebellum, telangiectasia, a defective immune system, and susceptibility to malignant tumors (especially leukemia and lymphoma). Chromosomes are often broken and inverted. Chromosome 7 and chromosome 14 are often translocated. Other symptoms include premature aging and endocrine abnormalities. AT cells are more likely to die than normal cells when exposed to ionizing radiation. ⁇ Genome Res 1997 Jim; 7 (6): 592- 605 ⁇
  • Ataxia-capillary vasodilation is caused by mutations in ataxia-capillary telangiectasia mutation gene (ATM, mutated in A-T). This gene is located on chromosomes llq22-q23. ATM gene products play a role in the regulation of the cell cycle and also play a role in the response to DNA damage. A-T heterozygotes are susceptible to tumors, especially breast cancer in women. [Cancer Res 1996 Sep 15; 56 (18): 4130-3]
  • the human ataxia-telangiectasia mutant protein 15 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, so there has been a need in the art. Identify more people involved in these processes ataxia-capillary Angiotensin mutein 15 protein, especially the amino acid sequence of this protein is identified. Isolation of the ataxia-capillary telangiectasia mutant protein 15 protein 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 agents for disease 1 and it is therefore important to isolate its coding DNA. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human ataxia-capillary telangiectasia mutant protein 15.
  • Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human ataxia-capillary telangiectasia mutant protein 15.
  • Another object of the present invention is to provide a method for producing human ataxia-capillary telangiectasia mutant protein 15.
  • Another object of the present invention is to provide an antibody against the human ataxia-capillary telangiectasia mutant protein 15 of the polypeptide of the present invention.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the ataxia-capillary telangiectasia mutant protein 15 of the polypeptide of the present invention.
  • Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of human ataxia-telangiectasia mutant protein 15.
  • 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:
  • (C) A polynucleotide having at least 70% identity to the polynucleotide sequence of (a) or (b).
  • sequence of the polynucleotide is one selected from the group consisting of: (a) having SEQ ID D NO: 1
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; a package
  • the method of preparing the polypeptide of the present invention includes culturing the host cell and recovering the expressed product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit human ataxia-capillary telangiectasia mutein 15 protein activity, which comprises utilizing the polypeptide of the invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for detecting a disease or disease susceptibility associated with abnormal expression of human ataxia-capillary telangiectasia mutant protein 15 in vitro, which comprises detecting the polypeptide or a polynucleotide sequence encoding the same Mutations, or the amount or biological activity of a polypeptide of the invention in a biological sample.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a polypeptide of the invention or a mimetic thereof, an activator, an antagonist or an inhibitor, and a pharmaceutically acceptable carrier.
  • the present invention also relates to the polypeptides and / or polynucleotides of the present invention prepared for the treatment of ataxia-capillary telangiectasia, breast cancer, various tumors, embryonic developmental disorders, growth disorders, immune diseases, Use of inflammation, hematopathy, HIV infection or other diseases caused by abnormal expression of human ataxia-telangiectasia mutant protein 15.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DM or 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 “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 means the replacement of a different amino acid or A nucleotide replaces one or more amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.
  • An "agonist” refers to a molecule that, when combined with human ataxia-capillary telangiectasia mutant protein 15 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 binds to human ataxia-capillary telangiectasia mutant protein 15.
  • Antagonist refers to an organism that can block or regulate human ataxia-capillary telangiectasia mutant protein 15 when combined with human ataxia-capillary telangiectasia mutant protein 15 Molecularly active or immunologically active molecule.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to human ataxia-capillary telangiectasia mutant protein 15.
  • Regular refers to a change in the function of human ataxia-capillary telangiectasia mutant protein 15 including an increase or decrease in protein activity, changes in binding properties, and human ataxia-capillary telangiectasia mutant protein 15. Any other biological, functional or immune change.
  • substantially pure ' means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated.
  • Those skilled in the art can purify human ataxia-capillary vasodilation using standard protein purification techniques.
  • Mutein 15 Essentially pure human ataxia-capillary telangiectasia mutant protein 15 produces a single main band on a non-reducing polyacrylamide gel. Human ataxia-capillary telangiectasia mutant protein 15 peptide The purity 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
  • complementary sequence G-A-C-T.
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. This inhibition of hybridization can be detected by performing hybridization (Sout hern 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 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. Percent identity can be determined electronically, such as through the MEGAU GN program (Lasergene software package, DNASTAR, Inc., Mad son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Cluster method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). The Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups.
  • sequence A and sequence B The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence X 100 The number of residues in sequence A-the number of spacer residues in sequence A Number of interval residues in a sequence B
  • the percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art such as Jotun Hein (Hein J., (1990) Methods in erazumology 183: 625-645).
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DNA or RNA sequence.
  • Antisense strand refers to a nucleic acid strand that is complementary to a “sense strand.”
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? It can specifically bind to the epitope of human ataxia-telangiectasia mutant protein 15.
  • a “humanized antibody” refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
  • isolated refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist 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 a component of its natural environment, they are still isolated.
  • isolated means that the substance is separated from its original environment (if it is natural Natural material, the original environment is the natural environment).
  • natural Natural material the original environment is the natural environment.
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .
  • isolated human ataxia-capillary telangiectasia mutant protein 15 refers to human ataxia-capillary telangiectasia mutant protein 15 which is substantially free of other proteins, lipids, and sugars naturally associated with it Or other substances.
  • Those skilled in the art can purify human ataxia-telangiectasia mutant protein 15 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human ataxia-capillary telangiectasia mutant protein 15 can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, ataxia-capillary telangiectasia mutant protein 15, 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 may be produced from a prokaryotic or eukaryotic host (for example, 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 ataxia-capillary telangiectasia mutant protein 15.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human ataxia-capillary telangiectasia mutant protein 15 of the present invention.
  • a fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution
  • the amino acid may or may not be encoded by a genetic codon; or (II) such a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (in) such One, wherein the mature polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide ( Such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence)
  • an additional amino acid sequence is fused into the mature polypeptide (such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or
  • 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.
  • Polynucleotides of the invention are found from a CDM library of human fetal brain tissue. It contains a polynucleotide sequence of 1462 bases in length and its open reading frame 1007-1258 encodes 139 amino acids.
  • this peptide is ataxia-capillary dilatation with human
  • the protein product of the mutated mutant gene has a similar expression profile, and it can be deduced that the human ataxia-capillary telangiectasia mutant protein 15 has similar functions to the protein product of the human ataxia-capillary telangiectasia mutant gene.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DM.
  • MA 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); 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 ) Add a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% F i co ll, 42 ° C, etc .; or (3) only between the two sequences Crosses occur at least 95% or more, and more preferably 97% or more.
  • the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • the "acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nucleotides. Nucleic acid fragments It can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding human ataxia-capillary telangiectasia mutant protein 15. The 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 ataxia-telangiectasia mutant protein 15 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments.
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the leg 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.
  • 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 of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-MA or DNA-RNA hybridization; (2) the presence or absence of a marker gene function; (3) determination of transcription of human ataxia-capillary telangiectasia mutant protein 15 (4) Detecting protein products expressed by genes through immunological techniques or measuring biological activity. The above methods can be used alone or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention.
  • the genes or fragments of the present invention can of course be used as probes.
  • DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
  • the protein product of human ataxia-telangiectasia mutant protein 15 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (EL ISA). Wait.
  • immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (EL ISA). Wait.
  • a method of amplifying DNA / 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 can be appropriately selected based on the polynucleotide sequence information of the present invention disclosed herein, and can be synthesized by conventional methods.
  • the amplified DM / RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DM fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell that is genetically engineered using the vector of the present invention or directly using the human ataxia-capillary telangiectasia mutant protein 15 coding sequence, and the recombinant technology to produce A method of inventing the polypeptide.
  • a polynucleotide sequence encoding human ataxia-capillary vasodilatation mutant protein 15 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al.
  • any plasmid and vector can be used to construct a recombinant expression vector.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
  • Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human ataxia-capillary telangiectasia mutant protein 15 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant MA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, 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 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 on the late side of the origin of replication, late on the origin of replication Polyoma enhancer and adenovirus enhancer on one side.
  • 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 ataxia-capillary vasodilation mutant protein 15 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a gene containing the polynucleotide or the recombinant vector.
  • Engineered host cells refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as fly S2 or Sf 9
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a 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 DNA can be harvested after the exponential growth phase and treated with CaCl, the steps used are well known in the art. The alternative is to use MgC l 2 .
  • 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 ataxia-capillary telangiectasia mutant protein 15 (Scence, 1984; 224: 1431). Generally there are the following steps:
  • polynucleotide or variant
  • encoding human ataxia-telangiectasia mutant protein 15 of the present invention or a suitable host transformed or transduced with a recombinant expression vector containing the polynucleotide Cell
  • 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, it can be separated by various separation methods using its physical, chemical and other properties. Isolate and purify the recombinant protein. 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
  • FIG. 1 is a comparison diagram of gene chip expression profiles of the protein product of the ataxia-capillary telangiectasia mutant protein 15 and the human ataxia-capillary telangiectasia mutant gene of the present invention.
  • the upper figure is a graph of the expression profile of the human ataxia-capillary vasodilation mutant protein 15.
  • the lower sequence is the graph of the expression profile of the protein product of the human ataxia-capillary vasodilation mutant gene.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human ataxia-telangiectasia mutant protein 15.
  • 15KDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform.
  • Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolation Kit (Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA. Use Smart cDNA Cloning Kit (purchased from Clontech). The 0 ⁇ fragment was inserted into the multiple cloning site of the pBSK (+) vector (Clontech), and transformed into DH5 ⁇ . The bacteria formed a cDNA library.
  • Dye terminate cycle react ion sequencing kit Perkin-Elmer
  • ABI 377 automatic sequencer Perkin-Eimer
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones, 062b09, was new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • Example 2 Cloning of the gene encoding human ataxia-capillary telangiectasia mutant protein 15 by RT-PCR method Using fetal brain cell total RNA as a template and oligo-dT as a primer for reverse transcription reaction to synthesize cDNA.
  • PCR amplification was performed with the following primers:
  • Primerl 5,-GAATGTGGTCATGTCATTTCTAGA -3, (SEQ ID NO: 3)
  • Priraer2 5,-AAAGCCAACATTTATTATATGTAC -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 'end reverse sequence in SEQ ID NO: 1.
  • Amplification conditions 50 ⁇ l of KC1, 10mniol / L Tris-CI, (pH8.5), 1.5mmol / L MgCl 2 , 200 ⁇ mol / L dNTP, lOpmol in a reaction volume of 50 ⁇ 1 Primer, 1U Taq DNA polymerase (Clontech).
  • the reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min.
  • RT-PCR (3-act in is the positive control and the template blank is the negative control.
  • the amplified product is purified using QIAGEN's kit, and the TA cloning kit is connected to the PCR vector (Invitrogen's product).
  • DNA The sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1 to 1462bp shown in SEQ ID NO: 1.
  • Example 3 Northern blot analysis of human ataxia-capillary telangiectasia mutant protein 15 gene expression:
  • RNA extraction using a one-step method includes acid guanidinium thiocyanate phenol-chloroform extraction. 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M acetic acid Sodium (pH4.0) was used to homogenize the tissue, 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) were added, and the mixture was centrifuged. The aqueous phase layer was aspirated and isopropyl alcohol (0.8 Volume) and the mixture was centrifuged to obtain an RNA pellet. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • the DNA probe used was the PCR amplified human ataxia-capillary telangiectasia mutant protein 15 coding region sequence (1007bp to 1258bp) shown in Figure 1.
  • a 32P-labeled probe (about 2 x 10 6 c P m / ral) 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 ⁇ SSC-5 ⁇ Denhardt's solution and 200 ⁇ g / ml salmon sperm DNA. After hybridization, the filter was washed in 1 X SSC-0.1 ° /. SDS at 55 ° C 30 min. Phosphor Imager was then used for analysis and quantification.
  • Example 4 In vitro expression, isolation and purification of recombinant human ataxia-telangiectasia mutant protein 15 According to SEQ ID NO: 1 and the coding region sequence shown in FIG. 1, a pair of specific amplification primers was designed, The sequence is as follows:
  • Primer 3 5'- CCCCATATGATGGGAACTGGTTATTTTATTTTC —3, (Seq ID No: 5)
  • Primer4 5 a CCCGAATTCTTATCTTGAAGAGAGAAAACTG-3, (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI digestion sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene, respectively.
  • the Ndel and BamH I restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET-28 b (+) (Novagen product, Cat. No. 69865.3).
  • the PCR reaction was performed using the pBS-0062b09 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions are as follows: a total volume of 50 ⁇ l contains 10 pg of pBS- 0062b09 plasmid, Primer-3 and Primer-4, and 1 J is lOpmol, 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 BamHI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into the colibacillus DH5CC using the calcium chloride method.
  • Polypeptide synthesizer (product of PE company) was used to synthesize the following human ataxia-telangiectasia mutant protein 15 specific peptides:
  • 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 identified whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected.
  • the probe can also be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissues or Whether the expression in pathological 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 imprinting, Nor thern blotting, and copying methods. They all use the same basic hybridization method 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 to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer.
  • the pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid.
  • unhybridized probes are removed by a series of membrane washes.
  • This embodiment uses higher-intensity washing conditions (such as lower salt concentration and higher temperature) to reduce the hybridization background and retain only strong specific signals.
  • the probes used in this embodiment include two types: the first type of probes are oligonucleotide fragments that are completely the same as or complementary to the polynucleotide SEQ ID NO: 1 of the present invention; the second type of probes are partially related to the present invention
  • the polynucleotide SEQ ID NO: 1 is the same or complementary oligonucleotide fragment.
  • the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
  • oligonucleotide fragments 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
  • the GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
  • 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 The column and its complementary region 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 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 mutant sequence of the gene fragment of SEQ ID NO: 1 or its complementary fragment (41Nt):
  • PBS phosphate buffered saline
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • NC membranes nitrocellulose membranes
  • Two NC membranes are required for each probe for subsequent experiments.
  • the film is washed with high-strength conditions and strength conditions, respectively.
  • prehybridization solution 10xDenhardfs; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DM).
  • Example 7 DNA Microarray Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases . The specific method steps have been reported in the literature.
  • a total of 4,000 polynucleotide sequences of different full-length cDNAs were prepared as the target DM, including the polynucleotide of the present invention. They were amplified by PCR respectively. After purification, the amplified product was adjusted to a concentration of about 500 ng / ul, and spotted on a glass medium using a Cartesian 7500 spotter (purchased from Cartesian, USA). The distance is 280 ⁇ !. The spotted slides were hydrated, dried, and cross-linked in a purple diplomatic coupling instrument. After elution, the DNA was fixed on a glass slide to prepare a chip.
  • the specific method steps have been variously reported in the literature, and the post-spotting processing steps of this embodiment are:
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and mRNA was purified with Oligotex mRNA Midi Kit (purchased from QiaGen).
  • the fluorescent reagent Cy3dUTP 5- Amino- propargy 2'- deoxyuridine 5'- triphate cou led to Cy3 fluorescent dye, purchased from Amersham Phamacia Biotech
  • Cy5dUTP 5- Amino- propargyl- 2'- deoxyur idine 5'-tr iphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech company, labeled the body's specific tissue (or stimulated cell line) mRNA, and purified the probe to prepare a probe.
  • the above specific tissues are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA- Ecv304 cell line, non-starved L02 cell line, Arsenic stimulated the L02 cell line and prostate tissue for 1 hour.
  • polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.
  • Ataxia-Capillary telangiectasia is an autosomal recessive disorder with symptoms such as ataxia of the cerebellum, telangiectasia, defective immune system, and susceptibility to malignant tumors (especially leukemia and lymphoma).
  • Ataxia-Capillary vasodilation is caused by ataxia-Capillary telangiectasia mutation (ATM, mutated in A-T) mutation.
  • ATM gene products play a role in the regulation of the cell cycle and also play a role in the response to DNA damage.
  • A-T heterozygotes are susceptible to tumors, especially breast cancer in women.
  • the expression profile of the polypeptide of the present invention is consistent with the expression profile of the protein product of the human ataxia-telangiectasia mutant gene, and both have similar biological functions. It has a variety of important functions in the body, mainly regulating the physiological functions of various tissues and organs in the body such as the immune system, cell proliferation, cerebellum, capillaries, etc. Abnormal expression will cause abnormalities in the above-mentioned tissue cells and cause related diseases.
  • the abnormal expression of the human ataxia-capillary telangiectasia mutant protein 15 will produce various diseases, especially ataxia-capillary telangiectasia, breast cancer, various tumors, embryonic developmental disorders, Growth disorders, immune diseases, inflammation, these diseases include but are not limited to: Tumors of various tissues: breast cancer, stomach cancer, liver cancer, lung cancer, esophageal cancer, leukemia, lymphoma, thyroid tumors, 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, tracheal tumor, fibroid, fibrosarcoma, lipoma, liposarcoma, embryonic developmental disorders Symptoms: Congenital abortion,
  • 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
  • Immune diseases common variable immunodeficiency disease, primary B lymphocyte immunodeficiency disease, primary T lymphocyte immunodeficiency disease, acquired immunodeficiency syndrome, systemic lupus erythematosus, rheumatoid arthritis,
  • Inflammation chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations
  • the abnormal expression of the human ataxia-telangiectasia mutant protein 15 of the present invention will also produce certain hereditary, hematological diseases and the like.
  • the polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially ataxia-capillary vasodilation, breast cancer, various tumors, and embryo development. Disorders, growth disorders, immune disorders, inflammation, certain hereditary, hematological diseases, etc.
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human ataxia-capillary telangiectasia mutant protein 15. Agonists enhance human ataxia-capillary vasodilator mutant protein 15 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers.
  • a mammalian cell or a membrane preparation expressing human ataxia-capillary telangiectasia mutant protein 15 can be cultured with a labeled human ataxia-capillary telangiectasia mutant protein 15 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human ataxia-telangiectasia mutant protein 15 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonist of human ataxia-capillary telangiectasia mutant protein 15 can bind to human ataxia-capillary telangiectasia mutant protein 15 and eliminate its function, or inhibit the production of the polypeptide, or with the polypeptide Binding of the active site prevents the polypeptide from performing biological functions can.
  • human ataxia-capillary vasodilatation mutant protein 15 can be added to the bioanalytical assay. The effect of this interaction is used to determine whether the 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 ataxia-capillary telangiectasia mutant protein 15 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In screening, 15 molecules of ataxia-telangiectasia mutant protein should generally be labeled.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies directed against the human ataxia-capillary telangiectasia mutant protein 15 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Polyclonal antibodies can be produced by direct injection of human ataxia-capillary telangiectasia mutant protein 15 into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but It is not limited to Freund's adjuvant and the like.
  • Techniques for preparing monoclonal antibodies to human ataxia-telangiectasia mutant protein 15 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta cells Hybridoma technology, EBV-hybridoma technology, etc.
  • Chimeric antibodies that bind human constant regions and non-human-derived variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851).
  • the existing technology for producing single-chain antibodies (U.S. Pat No. 4946778) can also be used to produce single-chain antibodies against human ataxia-capillary telangiectasia mutant protein 15.
  • Anti-human ataxia-telangiectasia mutant protein 15 antibody can be used in immunohistochemical techniques to detect human ataxia-telangiectasia mutant protein 15 in biopsy specimens.
  • Monoclonal antibodies that bind to human ataxia-telangiectasia mutant protein 15 can also be labeled with radioactive isotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • High-affinity monoclonal antibodies can covalently bind to bacterial or phytotoxins (such as diphtheria toxin, ricin, ormosine, etc.).
  • a common method is to attack the amino group of an antibody with a thiol crosslinker 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 ataxia-capillary vasodilation Mutein 15 positive cells.
  • the antibodies in the present invention can be used to treat or prevent ataxia-capillary telangiectasia mutant eggs White 15 related diseases. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human ataxia-telangiectasia mutant protein 15.
  • the present invention also relates to a diagnostic test method for quantitatively and locally detecting human ataxia-capillary vasodilation mutant protein level 15.
  • tests are well known in the art and include FI SH assays and radioimmunoassays.
  • Human ataxia-capillary telangiectasia mutant protein 15 levels detected in the test can be used to explain the importance of human ataxia-capillary telangiectasia mutant protein 15 in various diseases and to diagnose human ataxia Dysregulation-A disease in which telangiectasia mutant protein 15 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 ataxia-capillary telangiectasia mutant protein 15 can also be used for a variety of therapeutic purposes. Gene therapy techniques can be used to treat abnormalities in cell proliferation, development, or metabolism caused by human ataxia-capillary telangiectasia mutant protein 15 expression or abnormal / inactive expression. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutant human ataxia-capillary telangiectasia mutant protein 15 to inhibit endogenous human ataxia-capillary telangiectasia mutant protein 1 5 active.
  • a variant human ataxia-telangiectasia mutant protein 15 may be a shortened human ataxia-telangiectasia mutant protein 15 that lacks a signaling domain, although it may interact with downstream substrates. Binding, but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of ataxia-telangiectasia mutant protein 15.
  • 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 ataxia-capillary telangiectasia mutant protein 15 into cells .
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding a human ataxia-capillary telangiectasia mutant protein 15 can be found in the existing literature (Sambrook, et al.).
  • a polynucleotide encoding human ataxia-capillary telangiectasia mutant protein 15 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 DM
  • ribozymes that inhibit human ataxia-telangiectasia mutant protein 15 mRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RM molecule that can specifically decompose a specific A. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis has been widely used.
  • the daughter can be obtained by in vitro or in vivo transcription of the DNA sequence encoding the RM.
  • This DNA sequence has been integrated downstream of the RM polymerase promoter of the vector.
  • the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • a polynucleotide encoding human ataxia-capillary telangiectasia mutant protein 15 can be used for diagnosis of diseases related to human ataxia-capillary telangiectasia mutant protein 15.
  • a polynucleotide encoding human ataxia-capillary telangiectasia mutant protein 15 can be used to detect the expression of human ataxia-capillary telangiectasia mutant protein 15 or human ataxia-capillary telangiectasia in a disease state Aberrant Expression of Mutant Protein 1-5.
  • a DNA sequence encoding human ataxia-capillary telangiectasia mutant protein 15 can be used to hybridize biopsy specimens to determine the expression of human ataxia-capillary telangiectasia mutant protein 15.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available.
  • Some or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human ataxia-telangiectasia mutant protein 15 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect the human ataxia-telangiectasia mutant protein 15 transcription product.
  • Human ataxia-capillary telangiectasia mutant protein 15 mutations can also be used to diagnose human ataxia-capillary telangiectasia mutant protein 15-related diseases.
  • Human ataxia-capillary telangiectasia mutant protein 15 mutant forms include point mutations, translocations, deletions, recombinations and any other mutations compared to normal wild-type human ataxia-capillary telangiectasia mutant protein 1 5 DNA sequences Exception, etc. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, the Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • the sequences of the invention are also valuable for chromosome identification.
  • the sequence specifically targets a specific position on a human chromosome and can hybridize to it.
  • 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.
  • an important first step is to locate these DNA sequences on a chromosome.
  • a PCR primer (preferably 15-35bp) is prepared from the cDNA, and the sequence can be located on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes.
  • sublocalization can be achieved by a similar method using a set of fragments from a specific chromosome or a large number of genomic clones.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDNA libraries.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendel ian 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 cDNA or genomic sequence differences between the affected and the affected 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 diseased individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the 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 ataxia-telangiectasia mutant protein 15 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human ataxia-capillary vasodilator mutein 15 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

The present invention discloses a novel polypeptide - human ataxia-telangiectasia mutant protein 15 and a polynucleotide encoding the same, as well as a method of producing the polypeptide by DNArecombinant technique. The present invention also discloses methods of using the polypeptide in treatment of various disease, suchas ataxia-telangiectasia, breast cancer, various tumour, disturbance of embryo development, disorder of growth and development, immunological disease, inflammation, blood disease, HIV infection and so on. The present invention also discloses an antagonist against the polypeptide and the therapeutic use of the same. Also disclosed is the use of such novel polynucleotide encoding human ataxia-telangiectasia mutant protein 15.

Description

一种新的多肽一一人共济失调 -毛细血管扩张症突变蛋白 15  A New Peptide-Human Ataxia-Capillary Dilatation Mutant 15
和编码这种多肽的多核苷酸 技术领域  And a polynucleotide encoding such a polypeptide
本发明属于生物技术领域， 具体地说， 本发明描述了一种新的多肽一一人 共济失调 -毛细血管扩张症突变蛋白 15, 以及编码此多肽的多核苷酸序列。 本 发明还涉及此多核苷酸和多肽的制备方法和应用。 背景技术  The present invention belongs to the field of biotechnology, and specifically, the present invention describes a new polypeptide, ataxia-capillary telangiectasia mutant protein 15, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and polypeptide. Background technique
共济失调 -毛细血管扩张症 （ Ataxia- telangiectasia, AT)是一种常染色 体隐性失调。 其症状是小脑共济失调， 毛细血管扩张， 免疫系统缺陷、 易患恶 性肿瘤 （特别是白血病和淋巴瘤） 等。 染色体常有断裂、 倒位现象。 其 7 号染 色体和 14 号染色体经常有易位现象。 其他症状还包括早衰和内分泌异常。 AT 细胞在离子辐射的照射下比正常细胞更容易死亡。 【 Genome Res 1997 Jim; 7 (6): 592- 605】  Ataxia-telangiectasia (AT) is a recessive disorder of frequently chromosomal body. Symptoms are ataxia of the cerebellum, telangiectasia, a defective immune system, and susceptibility to malignant tumors (especially leukemia and lymphoma). Chromosomes are often broken and inverted. Chromosome 7 and chromosome 14 are often translocated. Other symptoms include premature aging and endocrine abnormalities. AT cells are more likely to die than normal cells when exposed to ionizing radiation. 【Genome Res 1997 Jim; 7 (6): 592- 605】
共济失调 -毛细血管扩张症是由于共济失调 -毛细血管扩张症突变基因 ( ATM, mutated in A-T ) 突变而引起的。 这一基因位于染色体 llq22- q23 位 置上。 ATM 基因产物在细胞周期的调节中起作用， 另外还在 DNA 损伤的反应中 起一定作用。 A-T的杂合子易患肿瘤， 特别是女性的乳腺癌。 [Cancer Res 1996 Sep 15; 56 (18)： 4130-3】  Ataxia-capillary vasodilation is caused by mutations in ataxia-capillary telangiectasia mutation gene (ATM, mutated in A-T). This gene is located on chromosomes llq22-q23. ATM gene products play a role in the regulation of the cell cycle and also play a role in the response to DNA damage. A-T heterozygotes are susceptible to tumors, especially breast cancer in women. [Cancer Res 1996 Sep 15; 56 (18): 4130-3]
到目前为止，已经发现了上百种 ATM位置的突变。其中有些突变可以减轻 A-T 症的症状。 ATM 基因的作用还不是很清楚， 但是很可能是保护细胞不受电离辐 射和其他拟辐射试剂的危害。 在组织培养的细胞中表达 ATM基因可以扭转 A - T 症易于受辐射影响的特点。 【Int J Radiat Biol 1998 Apr; 73 (4)： 365- 71】 通过基因芯片的分析发现， 在胸腺、 睾丸、 肌肉、 脾脏、 肺、 皮肤、 甲状 腺、 肝、 PMA+的 Ecv304 细胞株、 PMA -的 Ecv304 细胞株、 未饥饿的 L02 细胞 株、 砷刺激 1 小时的 L02 细胞株以及前列腺组织中， 本发明的多肽的表达谱与 人共济失调 -毛细血管扩张症突变基因的蛋白产物的表达谱非常近似， 因此二 者功能也可能类似。 本发明被命名为人共济失调 -毛细血管扩张症突变蛋白 15。  So far, mutations in hundreds of ATM positions have been discovered. Some of these mutations can reduce the symptoms of A-T. The role of the ATM gene is not yet clear, but it is likely to protect cells from ionizing radiation and other radiation-like agents. Expression of the ATM gene in tissue culture cells can reverse the susceptibility of A-T to radiation. [Int J Radiat Biol 1998 Apr; 73 (4): 365-71] Analysis of the gene chip revealed that in the thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA- In the Ecv304 cell line, the non-starved L02 cell line, the arsenic-stimulated L02 cell line, and the prostate tissue, the expression profile of the polypeptide of the present invention is very similar to that of the human ataxia-capillary telangiectasia mutant protein product. Approximately, so the functions of the two may be similar. The present invention is named human ataxia-capillary vasodilatation mutant protein 15.
由于如上所述人共济失调 -毛细血管扩张症突变蛋白 15 蛋白在调节细胞分 裂和胚胎发育等机体重要功能中起重要作用， 而且相信这些调节过程中涉及大 量的蛋白， 因而本领域中一直需要鉴定更多参与这些过程的人共济失调 -毛细 血管扩张症突变蛋白 1 5 蛋白， 特别是鉴定这种蛋白的氨基酸序列。 新人共济 失调 -毛细血管扩张症突变蛋白 15 蛋白编码基因的分离也为研究确定该蛋白 在健康和疾病状态下的作用提供了基础。 这种蛋白可能构成开发疾 1 病诊断和 /或治疗药的基础， 因此分离其编码 DNA是非常重要的。 发明的公开 As mentioned above, the human ataxia-telangiectasia mutant protein 15 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, so there has been a need in the art. Identify more people involved in these processes ataxia-capillary Angiotensin mutein 15 protein, especially the amino acid sequence of this protein is identified. Isolation of the ataxia-capillary telangiectasia mutant protein 15 protein 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 agents for disease 1 and it is therefore important to isolate its coding DNA. Disclosure of invention
本发明的一个目的是提供分离的新的多肽一一人共济失调 -毛细血管扩张 症突变蛋白 15 以及其片段、 类似物和衍生物。  It is an object of the present invention to provide isolated novel polypeptides-human ataxia-telangiectasia mutant protein 15 and fragments, analogs and derivatives thereof.
本发明的另一个目的是提供编码该多肽的多核苷酸。  Another object of the invention is to provide a polynucleotide encoding the polypeptide.
本发明的另一个目的是提供含有编码人共济失调 -毛细血管扩张症突变蛋 白 15的多核苷酸的重组载体。  Another object of the present invention is to provide a recombinant vector containing a polynucleotide encoding a human ataxia-capillary telangiectasia mutant protein 15.
本发明的另一个目的是提供含有编码人共济失调 -毛细血管扩张症突变蛋 白 1 5的多核苷酸的基因工程化宿主细胞。  Another object of the present invention is to provide a genetically engineered host cell containing a polynucleotide encoding a human ataxia-capillary telangiectasia mutant protein 15.
本发明的另一个目的是提供生产人共济失调 -毛细血管扩张症突变蛋白 1 5 的方法。  Another object of the present invention is to provide a method for producing human ataxia-capillary telangiectasia mutant protein 15.
本发明的另一个目的是提供针对本发明的多肽一一人共济失调 -毛细血管 扩张症突变蛋白 15的抗体。  Another object of the present invention is to provide an antibody against the human ataxia-capillary telangiectasia mutant protein 15 of the polypeptide of the present invention.
本发明的另一个目的是提供了针对本发明多肽一一人共济失调 -毛细血管 扩张症突变蛋白 1 5的模拟化合物、 拮抗剂、 激动剂、 抑制剂。  Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors of the ataxia-capillary telangiectasia mutant protein 15 of the polypeptide of the present invention.
本发明的另一个目的是提供诊断治疗与人共济失调 -毛细血管扩张症突变 蛋白 15异常相关的疾病的方法。  Another object of the present invention is to provide a method for diagnosing and treating diseases associated with abnormalities of human ataxia-telangiectasia mutant protein 15.
本发明涉及一种分离的多肽， 该多肽是人源的， 它包含： 具有 SEQ I D No. 2 氨基酸序列的多肽、 或其保守性变体、 生物活性片段或衍生物。 较佳地， 该多 肽是具有 SEQ I D NO: 2氨基酸序列的多肽。  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. Preferably, the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
本发明还涉及一种分离的多核苷酸， 它包含选自下组的一种核苷酸序列或 其变体：  The invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
(a)编码具有 SEQ I D No. 2氨基酸序列的多肽的多核苷酸；  (a) a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID D. 2;
(b)与多核苷酸（a)互补的多核苷酸；  (b) a polynucleotide complementary to polynucleotide (a);
(C )与（a)或（b)的多核苷酸序列具有至少 70%相同性的多核苷酸。  (C) A polynucleotide having at least 70% identity to the polynucleotide sequence of (a) or (b).
更佳地， 该多核苷酸的序列是选自下组的一种： （a)具有 SEQ I D NO: 1 中 More preferably, the sequence of the polynucleotide is one selected from the group consisting of: (a) having SEQ ID D NO: 1
1 007- 125 8位的序列； 和（b)具有 SEQ I D NO: 1中 1 -1462位的序列。 1 007- 125 8-bit sequence; and (b) a sequence having 1-1462 bits in SEQ ID NO: 1.
本发明另外涉及一种含有本发明多核苷酸的载体， 特别是表达载体； 一种 用该载体遗传工程化的宿主细胞， 包括转化、 转导或转染的宿主细胞； 一种包 括培养所述宿主细胞和回收表达产物的制备本发明多肽的方法。 The invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; a package The method of preparing the polypeptide of the present invention includes culturing the host cell and recovering the expressed product.
本发明还涉及一种能与本发明多肽特异性结合的抗体。  The invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
本发明还涉及一种筛选的模拟、 激活、 拮抗或抑制人共济失调 -毛细血管 扩张症突变蛋白 15蛋白活性的化合物的方法， 其包括利用本发明的多肽。 本发 明还涉及用该方法获得的化合物。  The invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit human ataxia-capillary telangiectasia mutein 15 protein activity, which comprises utilizing the polypeptide of the invention. The invention also relates to compounds obtained by this method.
本发明还涉及一种体外检测与人共济失调 -毛细血管扩张症突变蛋白 15蛋 白异常表达相关的疾病或疾病易感性的方法， 包括检测生物样品中所述多肽或其 编码多核苷酸序列中的突变， 或者检测生物样品中本发明多肽的量或生物活性。  The invention also relates to a method for detecting a disease or disease susceptibility associated with abnormal expression of human ataxia-capillary telangiectasia mutant protein 15 in vitro, which comprises detecting the polypeptide or a polynucleotide sequence encoding the same Mutations, or the amount or biological activity of a polypeptide of the invention in a biological sample.
本发明也涉及一种药物组合物， 它含有本发明多肽或其模拟物、 激活剂、 拮 抗剂或抑制剂以及药学上可接受的载体。  The invention also relates to 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.
本发明还涉及本发明的多肽和 /或多核苷酸在制备用于治疗共济失调 -毛 细血管扩张症、 乳腺癌、 各种肿瘤、 胚胎发育紊乱症、 生长发育障碍性疾病、 免疫性疾病、 炎症， 血液病， HI V 感染或其它由于人共济失调 -毛细血管扩张 症突变蛋白 15表达异常所引起疾病的药物的用途。  The present invention also relates to the polypeptides and / or polynucleotides of the present invention prepared for the treatment of ataxia-capillary telangiectasia, breast cancer, various tumors, embryonic developmental disorders, growth disorders, immune diseases, Use of inflammation, hematopathy, HIV infection or other diseases caused by abnormal expression of human ataxia-telangiectasia mutant protein 15.
本发明的其它方面由于本文的技术的公开， 对本领域的技术人员而言是显而 易见的。 本说明书和权利要求书中使用的下列术语除非特别说明具有如下的含义： "核酸序列" 是指寡核苷酸、 核苷酸或多核苷酸及其片段或部分， 也可以 指基因组或合成的 DM或 RNA， 它们可以是单链或双链的， 代表有义链或反义链。 类似地， 术语 "氨基酸序列" 是指寡肽、 肽、 多肽或蛋白质序列及其片段或部 分。 当本发明中的 "氨基酸序列" 涉及一种天然存在的蛋白质分子的氨基酸序 列时， 这种 "多肽" 或 "蛋白质" 不意味着将氨基酸序列限制为与所述蛋白质 分子相关的完整的天然氨基酸。  Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein. The following terms used in this specification and claims have the following meanings unless specifically stated: "Nucleic acid sequence" refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DM or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand. Similarly, the term "amino acid sequence" refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof. When the "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 .
蛋白质或多核苷酸 "变体" 是指一种具有一个或多个氨基酸或核苷酸改变 的氨基酸序列或编码它的多核苷酸序列。 所述改变可包括氨基酸序列或核苷酸 序列中氨基酸或核苷酸的缺失、 插入或替换。 变体可具有 "保守性" 改变， 其 中替换的氨基酸具有与原氨基酸相类似的结构或化学性质， 如用亮氨酸替换异 亮氨酸。 变体也可具有非保守性改变， 如用色氨酸替换甘氨酸。  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.
"缺失" 是指在氨基酸序列或核苷酸序列中一个或多个氨基酸或核苷酸的 缺失。  "Deletion" refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
"插入" 或 "添加" 是指在氨基酸序列或核苷酸序列中的改变导致与天然存在 的分子相比， 一个或多个氨基酸或核苷酸的增加。 "替换" 是指由不同的氨基酸或 核苷酸替换一个或多个氨基酸或核苷酸。 "Insertion" or "addition" 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" means the replacement of a different amino acid or A nucleotide replaces one or more amino acids or nucleotides.
"生物活性" 是指具有天然分子的结构、 调控或生物化学功能的蛋白质。 类似 地， 术语 "免疫学活性" 是指天然的、 重组的或合成蛋白质及其片段在合适的动 物或细胞中诱导特定免疫反应以及与特异性抗体结合的能力。  "Biological activity" refers to a protein that has the structure, regulation, or biochemical function of a natural molecule. Similarly, the term "immunologically active" refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response in appropriate animals or cells and to bind to specific antibodies.
"激动剂" 是指当与人共济失调 -毛细血管扩张症突变蛋白 1 5结合时， 一 种可引起该蛋白质改变从而调节该蛋白质活性的分子。 激动剂可以包括蛋白 质、 核酸、 碳水化合物或任何其它可结合人共济失调 -毛细血管扩张症突变蛋 白 1 5的分子。  An "agonist" refers to a molecule that, when combined with human ataxia-capillary telangiectasia mutant protein 15 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 binds to human ataxia-capillary telangiectasia mutant protein 15.
"拮抗剂" 或 "抑制物" 是指当与人共济失调 -毛细血管扩张症突变蛋白 1 5 结合时， 一种可封闭或调节人共济失调 -毛细血管扩张症突变蛋白 1 5的生物学 活性或免疫学活性的分子。 拮抗剂和抑制物可以包括蛋白质、 核酸、 碳水化合 物或任何其它可结合人共济失调 -毛细血管扩张症突变蛋白 1 5的分子。  "Antagonist" or "inhibitor" refers to an organism that can block or regulate human ataxia-capillary telangiectasia mutant protein 15 when combined with human ataxia-capillary telangiectasia mutant protein 15 Molecularly active or immunologically active molecule. Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates or any other molecule that can bind to human ataxia-capillary telangiectasia mutant protein 15.
"调节" 是指人共济失调 -毛细血管扩张症突变蛋白 1 5的功能发生改变， 包括蛋白质活性的升高或降低、 结合特性的改变及人共济失调 -毛细血管扩张 症突变蛋白 15的任何其它生物学性质、 功能或免疫性质的改变。  "Regulation" refers to a change in the function of human ataxia-capillary telangiectasia mutant protein 15 including an increase or decrease in protein activity, changes in binding properties, and human ataxia-capillary telangiectasia mutant protein 15. Any other biological, functional or immune change.
"基本上纯' '是指基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。 本领域的技术人员能用标准的蛋白质纯化技术纯化人共济失调 -毛细血管扩张症 突变蛋白 15。 基本上纯的人共济失调 -毛细血管扩张症突变蛋白 15在非还原性聚 丙烯酰胺凝胶上能产生单一的主带。 人共济失调 -毛细血管扩张症突变蛋白 15 多 肽的纯度可用氨基酸序列分析。  "Substantially pure '" means substantially free of other proteins, lipids, sugars or other substances with which it is naturally associated. Those skilled in the art can purify human ataxia-capillary vasodilation using standard protein purification techniques. Mutein 15. Essentially pure human ataxia-capillary telangiectasia mutant protein 15 produces a single main band on a non-reducing polyacrylamide gel. Human ataxia-capillary telangiectasia mutant protein 15 peptide The purity can be analyzed by amino acid sequence.
"互补的" 或 "互补" 是指在允许的盐浓度和温度条件下通过碱基配对的 多核苷酸天然结合。 例如， 序列 "C- T- G- A" 可与互补的序列 "G-A-C- T" 结合。 两个单链分子之间的互补可以是部分的或全部的。 核酸链之间的互补程度对于 核酸链之间杂交的效率及强度有明显影响。  "Complementary" or "complementary" refers to the natural binding of polynucleotides by base-pairing under conditions of acceptable salt concentration and temperature. For example, the sequence "C-T-G-A" can be combined with the complementary sequence "G-A-C-T". The complementarity between two single-stranded molecules may be partial or complete. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands.
"同源性" 是指互补的程度， 可以是部分同源或完全同源。 "部分同源" 是指一种部分互补的序列， 其至少可部分抑制完全互补的序列与靶核酸的杂 交。 这种杂交的抑制可通过在严格性程度降低的条件下进行杂交 （Sout hern印 迹或 Nor thern印迹等） 来检测。 基本上同源的序列或杂交探针可竟争和抑制完 全同源的序列与靶序列在的严格性程度降低的条件下的结合。 这并不意味严格 性程度降低的条件允许非特异性结合， 因为严格性程度降低的条件要求两条序 列相互的结合为特异性或选择性相互作用。  "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 (Sout hern 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 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.
"相同性百分率" 是指在两种或多种氨基酸或核酸序列比较中序列相同或 相似的百分率。 可用电子方法测定相同性百分率， 如通过 MEGAU GN程序 ( Lasergene software package, DNASTAR, Inc. , Mad i son Wis. ) 。 MEGALIGN 程序可根据不同的方法如 Cluster法比较两种或多种序列（Higgins, D. G. 和 P.M. Sharp (1988) Gene 73:237-244)。 Clus ter法通过检查所有配对之间的 距离将各组序列排列成簇。 然后将各簇以成对或成组分配。 两个氨基酸序列如 序列 A和序列 B之间的相同性百分率通过下式计算： 序列 A与序列 B之间匹配的残基个数 X 100 序列 A的残基数一序列 A中间隔残基数一序列 B中间隔残基数 "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. Percent identity can be determined electronically, such as through the MEGAU GN program (Lasergene software package, DNASTAR, Inc., Mad son Wis.). The MEGALIGN program can compare two or more sequences according to different methods such as the Cluster method (Higgins, DG and PM Sharp (1988) Gene 73: 237-244). The Clus ter method arranges groups of sequences into clusters by checking the distance between all pairs. The clusters are then assigned in pairs or groups. The percent identity between two amino acid sequences such as sequence A and sequence B is calculated by the following formula: The number of matching residues between sequence A and sequence X 100 The number of residues in sequence A-the number of spacer residues in sequence A Number of interval residues in a sequence B
也可以通过 Cluster法或用本领域周知的方法如 Jotun Hein 测定核酸序列 之间的相同性百分率（Hein J.， (1990) Methods in erazumology 183: 625-645)。  The percent identity between nucleic acid sequences can also be determined by the Cluster method or by methods known in the art such as Jotun Hein (Hein J., (1990) Methods in erazumology 183: 625-645).
"相似性 " 是指氨基酸序列之间排列对比时相应位置氨基酸残基的相同或 保守性取代的程度。 用于保守性取代的氨基酸例如， 带负电荷的氨基酸可包括 天冬氨酸和谷氨酸； 带正电荷的氨基酸可包括赖氨酸和精氨酸； 具有不带电荷 的头部基团有相似亲水性的氨基酸可包括亮氨酸、 异亮氨酸和缬氨酸； 甘氨酸 和丙氨酸； 天冬酰胺和谷氨酰胺； 丝氨酸和苏氨酸； 苯丙氨酸和酪氨酸。  "Similarity" refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences. Amino acids used for conservative substitutions, for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
"反义" 是指与特定的 DNA或 RNA序列互补的核苷酸序列。 "反义链" 是指 与 "有义链" 互补的核酸链。  "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 a "sense strand."
"衍生物" 是指 HFP或编码其的核酸的化学修饰物。 这种化学修饰物可以是 用烷基、 酰基或氨基替换氢原子。 核酸衍生物可编码保留天然分子的主要生物 学特性的多肽。  "Derivative" refers to a chemical modification of HFP or a nucleic acid encoding it. This chemical modification may be the replacement of a hydrogen atom with an alkyl, acyl or amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological properties of natural molecules.
"抗体" 是指完整的抗体分子及其片段， 如 Fa、 ？(^')2及？ ， 其能特异 性结合人共济失调 -毛细血管扩张症突变蛋白 15的抗原决定簇。 "Antibody" refers to a complete antibody molecule and its fragments, such as Fa,? (^ ') 2 and? It can specifically bind to the epitope of human ataxia-telangiectasia mutant protein 15.
"人源化抗体" 是指非抗原结合区域的氨基酸序列被替换变得与人抗体更 为相似， 但仍保留原始结合活性的抗体。  A "humanized antibody" refers to an antibody in which the amino acid sequence of a non-antigen binding region is replaced to become more similar to a human antibody, but still retains the original binding activity.
"分离的" 一词指将物质从它原来的环境 （例如， 若是自然产生的就指其 天然环境） 之中移出。 比如说， 一个自然产生的多核苷酸或多肽存在于活动物 中就是没有被分离出来， 但同样的多核苷酸或多肽同一些或全部在自然系统中 与之共存的物质分开就是分离的。 这样的多核苷酸可能是某一载体的一部分， 也可能这样的多核苷酸或多肽是某一组合物的一部分。 既然载体或组合物不是 它天然环境的成分， 它们仍然是分离的。 如本发明所用， "分离的" 是指物质从其原始环境中分离出来 （如果是天 然的物质， 原始环境即是天然环境） 。 如活体细胞内的天然状态下的多聚核苷 酸和多肽是没有分离纯化的， 但同样的多聚核苷酸或多肽如从天然状态中同存 在的其他物质中分开， 则为分离纯化的。 The term "isolated" refers to the removal of a substance from its original environment (for example, its natural environment if it occurs naturally). For example, a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist 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 a component of its natural environment, they are still isolated. As used in the present invention, "isolated" means that the substance is separated from its original environment (if it is natural Natural material, the original environment is the natural environment). For example, polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .
如本文所用， "分离的人共济失调 -毛细血管扩张症突变蛋白 15" 是指人 共济失调 -毛细血管扩张症突变蛋白 15基本上不含天然与其相关的其它蛋白、 脂类、 糖类或其它物质。 本领域的技术人员能用标准的蛋白质纯化技术纯化人 共济失调 -毛细血管扩张症突变蛋白 15。 基本上纯的多肽在非还原聚丙烯酰胺 凝胶上能产生单一的主带。 人共济失调 -毛细血管扩张症突变蛋白 15 多肽的纯 度能用氨基酸序列分析。  As used herein, "isolated human ataxia-capillary telangiectasia mutant protein 15" refers to human ataxia-capillary telangiectasia mutant protein 15 which is substantially free of other proteins, lipids, and sugars naturally associated with it Or other substances. Those skilled in the art can purify human ataxia-telangiectasia mutant protein 15 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human ataxia-capillary telangiectasia mutant protein 15 can be analyzed by amino acid sequence.
本发明提供了一种新的多肽一一人共济失调 -毛细血管扩张症突变蛋白 15， 其基本上是由 SEQ ID NO: 2所示的氨基酸序列组成的。 本发明的多肽可以是重组多 肽、 天然多肽、 合成多肽， 优选重组多肽。 本发明的多肽可以是天然纯化的产物， 或是化学合成的产物， 或使用重组技术从原核或真核宿主（例如， 细菌、 酵母、 高 等植物、 昆虫和哺乳动物细胞）中产生。 根据重组生产方案所用的宿主， 本发明的 多肽可以是糖基化的， 或可以是非糖基化的。 本发明的多肽还可包括或不包括起 始的甲硫氨酸残基。  The present invention provides a new polypeptide, ataxia-capillary telangiectasia mutant protein 15, 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 may be produced from a prokaryotic or eukaryotic host (for example, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology. Depending on the host used in the recombinant production protocol, the 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.
本发明还包括人共济失调 -毛细血管扩张症突变蛋白 15 的片段、 衍生物 和类似物。 如本发明所用， 术语 "片段" 、 "衍生物" 和 "类似物" 是指基本 上保持本发明的人共济失调 -毛细血管扩张症突变蛋白 15 相同的生物学功能 或活性的多肽。 本发明多肽的片段、 衍生物或类似物可以是： （ I ) 这样一种， 其中一个或多个氨基酸残基被保守或非保守氨基酸残基 （优选的是保守氨基酸 残基） 取代， 并且取代的氨基酸可以是也可以不是由遗传密码子编码的； 或者 ( I I ) 这样一种， 其中一个或多个氨基酸残基上的某个基团被其它基团取代包 含取代基； 或者 （ i n ) 这样一种， 其中成熟多肽与另一种化合物 （ 比如延长 多肽半衰期的化合物， 例如聚乙二醇） 融合； 或者 （ IV ) 这样一种， 其中附加 的氨基酸序列融合进成熟多肽而形成的多肽序列 （如前导序列或分泌序列或用 来纯化此多肽的序列或蛋白原序列） 通过本文的阐述， 这样的片段、 00衍生物 和类似物被认为在本领域技术人员的知识范围之内。  The invention also includes fragments, derivatives and analogs of human ataxia-capillary telangiectasia mutant protein 15. As used in the present invention, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially maintains the same biological function or activity of the human ataxia-capillary telangiectasia mutant protein 15 of the present invention. A fragment, derivative or analog of the polypeptide of the present invention may be: (I) a kind in which one or more amino acid residues are substituted with conservative or non-conservative amino acid residues (preferably conservative amino acid residues), and the substitution The amino acid may or may not be encoded by a genetic codon; or (II) such a type in which a group on one or more amino acid residues is substituted by another group to include a substituent; or (in) such One, wherein the mature polypeptide is fused to another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (IV) such a polypeptide sequence in which an additional amino acid sequence is fused into the mature polypeptide ( Such as the leader sequence or secreted sequence or the sequence used to purify this polypeptide or protease sequence) As explained herein, such fragments, 00 derivatives and analogs are considered to be within the knowledge of those skilled in the art.
本发明提供了分离的核酸 （多核苷酸） ， 基本由编码具有 SEQ ID NO: 2 氨 基酸序列的多肽的多核苷酸组成。 本发明的多核苷酸序列包括 SEQ ID NO: 1 的 核苷酸序列。 本发明的多核苷酸是从人胎脑组织的 cDM 文库中发现的。 它包 含的多核苷酸序列全长为 1462 个碱基， 其开放读框 1007-1258 编码了 139 个 氨基酸。 根据基因芯片表达谱比较发现， 此多肽与人共济失调 -毛细血管扩张 症突变基因的蛋白产物有相似的表达谱， 可推断出该人共济失调 -毛细血管扩 张症突变蛋白 15 具有人共济失调 -毛细血管扩张症突变基因的蛋白产物相似 的功能。 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. Polynucleotides of the invention are found from a CDM library of human fetal brain tissue. It contains a polynucleotide sequence of 1462 bases in length and its open reading frame 1007-1258 encodes 139 amino acids. According to the comparison of gene chip expression profiles, this peptide is ataxia-capillary dilatation with human The protein product of the mutated mutant gene has a similar expression profile, and it can be deduced that the human ataxia-capillary telangiectasia mutant protein 15 has similar functions to the protein product of the human ataxia-capillary telangiectasia mutant gene.
本发明的多核苷酸可以是 DNA形式或是 RNA形式。 DNA形式包括 cDNA、 基 因组 DNA或人工合成的 DM。 MA可以是单链的或是双链的。 DNA可以是编码链 或非编码链。 编码成熟多肽的编码区序列可以与 SEQ I D NO: 1 所示的编码区序 列相同或者是简并的变异体。 如本发明所用， "简并的变异体" 在本发明中是 指编码具有 SEQ ID NO: 2 的蛋白质或多肽， 但与 SEQ ID NO: 1 所示的编码区序 列有差别的核酸序列。  The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DM. MA 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. As used herein, 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.
编码 SEQ ID N0: 2的成熟多肽的多核苷酸包括： 只有成熟多肽的编码序列； 成熟多肽的编码序列和各种附加编码序列； 成熟多肽的编码序列 （和任选的附 加编码序列） 以及非编码序列。  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.
术语 "编码多肽的多核苷酸" 是指包括编码此多肽的多核苷酸和包括附加 编码和 /或非编码序列的多核苷酸。  The term "polynucleotide encoding a polypeptide" refers to a polynucleotide comprising the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
本发明还涉及上述描述多核苷酸的变异体， 其编码与本发明有相同的氨基 酸序列的多肽或多肽的片断、 类似物和衍生物。 此多核苷酸的变异体可以是天 然发生的等位变异体或非天然发生的变异体。 这些核苷酸变异体包括取代变异 体、 缺失变异体和插入变异体。 如本领域所知的， 等位变异体是一个多核苷酸 的替换形式， 它可能是一个或多个核苷酸的取代、 缺失或插入， 但不会从实质 上改变其编码的多肽的功能。  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. As known in the art, 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 .
本发明还涉及与以上所描述的序列杂交的多核苷酸 （两个序列之间具有至 少 50% , 优选具有 70%的相同性） 。 本发明特别涉及在严格条件下与本发明所 述多核苷酸可杂交的多核苷酸。 在本发明中， "严格条件" 是指： （1)在较低 离子强度和较高温度下的杂交和洗脱， 如 0. 2xSSC, 0. 1%SDS, 60 °C ;或（2)杂交 时加用变性剂， 如 50% (v/v)甲酰胺， 0. 1%小牛血清 / 0. l%F i co l l， 42 °C等； 或（3) 仅在两条序列之间的相同性至少在 95%以上，更好是 97%以上时才发生杂交。 并 且， 可杂交的多核苷酸编码的多肽与 SEQ ID NO : 2 所示的成熟多肽有相同的 生物学功能和活性。 酸片段"的长度至少含 10个核苷酸， 较好是至少 20-30个核苷酸， 更好是至少 50-60 个核苷酸， 最好是至少 100 个核苷酸以上。 核酸片段也可用于核酸的扩 增技术（如 PCR)以确定和 /或分离编码人共济失调 -毛细血管扩张症突变蛋白 15 的多核苷酸。 本发明中的多肽和多核苷酸优选以分离的形式提供， 更佳地被纯化至均质。 本发明的编码人共济失调 -毛细血管扩张症突变蛋白 15的特异的多核苷酸 序列能用多种方法获得。 例如， 用本领域熟知的杂交技术分离多核苷酸。 这些 技术包括但不局限于： 1)用探针与基因组或 cDNA文库杂交以检出同源的多核苷 酸序列， 和 2)表达文库的抗体筛选以检出具有共同结构特征的克隆的多核苷酸 片段。 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. In the present invention, "strict conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 60 ° C; or ( 2 ) Add a denaturant during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% F i co ll, 42 ° C, etc .; or (3) only between the two sequences Crosses occur at least 95% or more, and more preferably 97% or more. In addition, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2. The "acid fragment" contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least 50-60 nucleotides, and most preferably at least 100 nucleotides. Nucleic acid fragments It can also be used in nucleic acid amplification techniques such as PCR to identify and / or isolate polynucleotides encoding human ataxia-capillary telangiectasia mutant protein 15. The 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 ataxia-telangiectasia mutant protein 15 of the present invention can be obtained by various methods. For example, 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.
本发明的 DNA片段序列也能用下列方法获得： 1)从基因组 DNA分离双链 DNA 序列； 2)化学合成腿序列以获得所述多肽的双链 DNA。  The DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the leg sequence to obtain the double-stranded DNA of the polypeptide.
上述提到的方法中， 分离基因组 DNA 最不常用。 DM 序列的直接化学合成 是经常选用的方法。 更经常选用的方法是 cDNA序列的分离。 分离感兴趣的 cDNA 的标准方法是从高表达该基因的供体细胞分离 mRNA并进行逆转录， 形成质粒或 噬菌体 cDNA 文库。 提取 mRNA 的方法已有多种成熟的技术， 试剂盒也可从商业 途径获得（Qiagene)。 而构建 cDNA 文库也是通常的方法（Sambrook, et al. , Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory. New York, 1989)。还可得到商业供应的 cDNA文库，如 Clontech公司的不同 cDNA 文库。 当结合使用聚合酶反应技术时， 即使极少的表达产物也能克隆。  Of the methods mentioned above, 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. 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.
可用常规方法从这些 cDNA 文库中筛选本发明的基因。 这些方法包括（但不 限于）： （l)DNA-MA 或 DNA-RNA 杂交； （2)标志基因功能的出现或丧失； （3)测 定人共济失调 -毛细血管扩张症突变蛋白 15 的转录本的水平； （4)通过免疫学 技术或测定生物学活性， 来检测基因表达的蛋白产物。 上述方法可单用， 也可 多种方法联合应用。  The genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-MA or DNA-RNA hybridization; (2) the presence or absence of a marker gene function; (3) determination of transcription of human ataxia-capillary telangiectasia mutant protein 15 (4) Detecting protein products expressed by genes through immunological techniques or measuring biological activity. The above methods can be used alone or in combination.
在第（1)种方法中， 杂交所用的探针是与本发明的多核苷酸的任何一部分同 源， 其长度至少 10个核苷酸， 较好是至少 30个核苷酸， 更好是至少 50个核苷 酸， 最好是至少 100个核苷酸。 此外， 探针的长度通常在 2000个核苷酸之内， 较佳的为 1000个核苷酸之内。 此处所用的探针通常是在本发明的基因序列信息 的基础上化学合成的 DNA序列。 本发明的基因本身或者片段当然可以用作探针。 DNA探针的标记可用放射性同位素， 荧光素或酶（如碱性磷酸酶）等。  In the method (1), 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. In addition, the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides. The probe used here is generally a DNA sequence chemically synthesized based on the gene sequence information of the present invention. The genes or fragments of the present invention can of course be used as probes. DNA probes can be labeled with radioisotopes, luciferin, or enzymes (such as alkaline phosphatase).
在第（4)种方法中， 检测人共济失调 -毛细血管扩张症突变蛋白 15 基因表 达的蛋白产物可用免疫学技术如 Western 印迹法， 放射免疫沉淀法， 酶联免疫 吸附法（EL ISA)等。  In the method (4), the protein product of human ataxia-telangiectasia mutant protein 15 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (EL ISA). Wait.
应 用 PCR 技 术 扩 增 DNA/RNA 的 方 法 （Saiki, et al. Science 1985; 230: 1350-1354)被优选用于获得本发明的基因。 特别是很难从文库中得到 全长的 cDNA 时， 可优选使用 RACE法（RACE - cDNA末端快速扩增法）， 用于 PCR 的引物可根据本文所公开的本发明的多核苷酸序列信息适当地选择， 并可用常 规方法合成。 可用常规方法如通过凝胶电泳分离和纯化扩增的 DM/RNA片段。 A method of amplifying DNA / RNA by PCR (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention. In particular, when it is difficult to obtain a full-length cDNA from a library, the RACE method (RACE-rapid amplification of cDNA ends) can be preferably used for PCR. The primers can be appropriately selected based on the polynucleotide sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The amplified DM / RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
如上所述得到的本发明的基因， 或者各种 DM 片段等的多核苷酸序列可用 常规方法如双脱氧链终止法（Sanger et al. PNAS, 1977 , 74： 5463-5467)测定。 这类多核苷酸序列测定也可用商业测序试剂盒等。为了获得全长的 cDNA序列， 测 序需反复进行。 有时需要测定多个克隆的 cDNA 序列， 才能拼接成全长的 cDNA 序列。  The polynucleotide sequence of the gene of the present invention or various DM fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
本发明也涉及包含本发明的多核苷酸的载体， 以及用本发明的载体或直接 用人共济失调 -毛细血管扩张症突变蛋白 15编码序列经基因工程产生的宿主细 胞， 以及经重组技术产生本发明所述多肽的方法。  The present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell that is genetically engineered using the vector of the present invention or directly using the human ataxia-capillary telangiectasia mutant protein 15 coding sequence, and the recombinant technology to produce A method of inventing the polypeptide.
本发明中， 编码人共济失调 -毛细血管扩张症突变蛋白 15的多核苷酸序列 可插入到载体中， 以构成含有本发明所述多核苷酸的重组载体。 术语 "载体" 指本领域熟知的细菌质粒、 噬菌体、 酵母质粒、 植物细胞病毒、 哺乳动物细胞 病毒如腺病毒、 逆转录病毒或其它载体。 在本发明中适用的载体包括但不限于： 在细菌中表达的基于 T7 启动子的表达载体（Rosenberg, et al. Gene, 1987, 56: 125)； 在哺乳动物细胞中表达的 pMSXND表达载体（Lee and Nathans, J Bio Chem. 263: 3521, 1988)和在昆虫细胞中表达的来源于杆状病毒的载体。 总之， 只要能在宿主体内复制和稳定， 任何质粒和载体都可以用于构建重组表达载体。 表达载体的一个重要特征是通常含有复制起始点、 启动子、 标记基因和翻译调 控元件。  In the present invention, a polynucleotide sequence encoding human ataxia-capillary vasodilatation mutant protein 15 can be inserted into a vector to constitute a recombinant vector containing the polynucleotide of the present invention. The term "vector" refers to bacterial plasmids, phages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors well known in the art. Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors (Rosenberg, et al. Gene, 1987, 56: 125) expressed in bacteria; pMSXND expression vectors expressed in mammalian cells ( Lee and Nathans, J Bio Chem. 263: 3521, 1988) and baculovirus-derived vectors expressed in insect cells. In short, as long as it can be replicated and stabilized in a host, any plasmid and vector can be used to construct a recombinant expression vector. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translational regulatory elements.
本领域的技术人员熟知的方法能用于构建含编码人共济失调 -毛细血管扩 张症突变蛋白 15 的 DNA 序列和合适的转录 /翻译调控元件的表达载体。 这些方 法包括体外重组 MA技术、 DNA合成技术、 体内重组技术等（Sambroook, et al. Molecular Cloning, a Laboratory Manual, cold Spring Harbor Laboratory. New York, 1989)。 所述的 DNA序列可有效连接到表达载体中的适当启动子上， 以指导 mRNA合成。 这些启动子的代表性例子有： 大肠杆菌的 lac或 trp启动子； λ噬菌体的 PL启动子； 真核启动子包括 CMV立即早期启动子、 HSV胸苷激酶启 动子、 早期和晚期 SV40 启动子、 反转录病毒的 LTRs 和其它一些已知的可控制 基因在原核细胞或真核细胞或其病毒中表达的启动子。 表达载体还包括翻译起 始用的核糖体结合位点和转录终止子等。 在载体中插入增强子序列将会使其在 高等真核细胞中的转录得到增强。 增强子是 DM 表达的顺式作用因子， 通常大 约有 10到 300个碱基对， 作用于启动子以增强基因的转录。 可举的例子包括在 复制起始点晚期一侧的 100到 270个碱基对的 SV40增强子、 在复制起始点晚期 一侧的多瘤增强子以及腺病毒增强子等。 Methods well known to those skilled in the art can be used to construct expression vectors containing a DNA sequence encoding human ataxia-capillary telangiectasia mutant protein 15 and appropriate transcription / translation regulatory elements. These methods include in vitro recombinant MA technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manual, 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. coli; the PL promoter of lambda phage; eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, Retroviral LTRs and other known promoters that control the expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also includes a ribosome binding site 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 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 on the late side of the origin of replication, late on the origin of replication Polyoma enhancer and adenovirus enhancer on one side.
此外， 表达载体优选地包含一个或多个选择性标记基因， 以提供用于选择 转化的宿主细胞的表型性状， 如真核细胞培养用的二氢叶酸还原酶、 新霉素抗 性以及绿色荧光蛋白（GFP) , 或用于大肠杆菌的四环素或氨苄青霉素抗性等。  In addition, 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.
本领域一般技术人员都清楚如何选择适当的载体 /转录调控元件 （如启动 子、 增强子等） 和选择性标记基因。  Those of ordinary skill in the art will know how to select appropriate vector / transcription control elements (such as promoters, enhancers, etc.) and selectable marker genes.
本发明中， 编码人共济失调 -毛细血管扩张症突变蛋白 15的多核苷酸或含 有该多核苷酸的重组载体可转化或转导入宿主细胞， 以构成含有该多核苷酸或 重组载体的基因工程化宿主细胞。 术语 "宿主细胞" 指原核细胞， 如细菌细胞； 或是低等真核细胞， 如酵母细胞； 或是高等真核细胞， 如哺乳动物细胞。 代表 性例子有： 大肠杆菌， 链霉菌属； 细菌细胞如鼠伤寒沙门氏菌； 真菌细胞如酵 母； 植物细胞； 昆虫细胞如果蝇 S2或 Sf 9 ; 动物细胞如 CH0、 COS或 Bowes 黑 素瘤细胞等。  In the present invention, a polynucleotide encoding human ataxia-capillary vasodilation mutant protein 15 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a gene containing the polynucleotide or the recombinant vector. Engineered host cells. The term "host cell" refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: E. coli, Streptomyces; bacterial cells such as Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells such as fly S2 or Sf 9; animal cells such as CH0, COS or Bowes melanoma cells.
用本发明所述的 DNA序列或含有所述 DNA序列的重组载体转化宿主细胞可 用本领域技术人员熟知的常规技术进行。 当宿主为原核生物如大肠杆菌时， 能 吸收 DNA 的感受态细胞可在指数生长期后收获， 用 CaC l ^ 处理， 所用的步骤 在本领域众所周知。 可供选择的是用 MgC l 2。 如果需要， 转化也可用电穿孔的方 法进行。 当宿主是真核生物， 可选用如下的 DNA 转染方法： 磷酸钙共沉淀法， 或者常规机械方法如显微注射、 电穿孔、 脂质体包装等。 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. When the host is a prokaryote such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl, the steps used are well known in the art. The alternative is to use MgC l 2 . If necessary, transformation can also be performed by electroporation. When 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.
通过常规的重组 DNA 技术， 利用本发明的多核苷酸序列可用来表达或生产 重组的人共济失调 -毛细血管扩张症突变蛋白 15 (Sc i ence , 1984 ; 224： 1431)。 一般来说有以下步骤：  Using conventional recombinant DNA technology, the polynucleotide sequence of the present invention can be used to express or produce recombinant human ataxia-capillary telangiectasia mutant protein 15 (Scence, 1984; 224: 1431). Generally there are the following steps:
(1) .用本发明的编码人 人共济失调 -毛细血管扩张症突变蛋白 15 的多核 苷酸（或变异体）， 或用含有该多核苷酸的重组表达载体转化或转导合适的宿主 细胞；  (1) The polynucleotide (or variant) encoding human ataxia-telangiectasia mutant protein 15 of the present invention, or a suitable host transformed or transduced with a recombinant expression vector containing the polynucleotide Cell
(2) .在合适的培养基中培养宿主细胞；  (2) culturing host cells in a suitable medium;
( 3) .从培养基或细胞中分离、 纯化蛋白质。  (3) Isolate and purify protein from culture medium or cells.
在步骤 （2 ) 中， 根据所用的宿主细胞， 培养中所用的培养基可选自各种 常规培养基。 在适于宿主细胞生长的条件下进行培养。 当宿主细胞生长到适当 的细胞密度后， 用合适的方法（如温度转换或化学诱导）诱导选择的启动子， 将 细胞再培养一段时间。  In step (2), depending on the host cell used, 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.
在歩骤 （ 3 ) 中， 重组多肽可包被于细胞内、 或在细胞膜上表达、 或分泌到 细胞外。 如果需要， 可利用其物理的、 化学的和其它特性通过各种分离方法分 离和纯化重组的蛋白。 这些方法是本领域技术人员所熟知的。 这些方法包括但 并不限于： 常规的复性处理、 蛋白沉淀剂处理（盐析方法）、 离心、 渗透破菌、 超声波处理、 超离心、 分子筛层析（凝胶过滤）、 吸附层析、 离子交换层析、 高 效液相层析（HPLC)和其它各种液相层析技术及这些方法的结合。 附图的简要说明 In step (3), the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell. If necessary, it can be separated by various separation methods using its physical, chemical and other properties. Isolate and purify the recombinant protein. 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. Brief description of the drawings
下列附图用于说明本发明的具体实施方案， 而不用于限定由权利要求书所 界定的本发明范围。  The following drawings are used to illustrate specific embodiments of the invention, but not to limit the scope of the invention as defined by the claims.
图 1是本发明人共济失调 -毛细血管扩张症突变蛋白 15和人共济失调 -毛 细血管扩张症突变基因的蛋白产物的基因芯片表达谱比较图。 上图是人共济失 调 -毛细血管扩张症突变蛋白 15的表达谱折方图， 下方序列是人共济失调 -毛 细血管扩张症突变基因的蛋白产物的表达谱折方图。  FIG. 1 is a comparison diagram of gene chip expression profiles of the protein product of the ataxia-capillary telangiectasia mutant protein 15 and the human ataxia-capillary telangiectasia mutant gene of the present invention. The upper figure is a graph of the expression profile of the human ataxia-capillary vasodilation mutant protein 15. The lower sequence is the graph of the expression profile of the protein product of the human ataxia-capillary vasodilation mutant gene.
图 2 为分离的人共济失调 -毛细血管扩张症突变蛋白 15 的聚丙烯酰胺凝 胶电泳图 （ SDS-PAGE) 。 15KDa 为蛋白质的分子量。 箭头所指为分离出的蛋白 条带。 实现本发明的最佳方式  Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of isolated human ataxia-telangiectasia mutant protein 15. 15KDa is the molecular weight of the protein. The arrow indicates the isolated protein band. The best way to implement the invention
下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说 明本发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方 法，通常按照常规条件如 Sambrook等人， 分子克隆：实验室手册（New York: Cold Spring Harbor Laboratory Press, 1989)中所述的条件， 或按照制造厂商所 建议的条件。  The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer Suggested conditions.
实施例 1: 人共济失调 -毛细血管扩张症突变蛋白 15的克隆  Example 1: Cloning of human ataxia-telangiectasia mutant protein 15
用异硫氰酸胍 /酚 /氯仿一步法提取人胎脑总 RNA。 用 Quik mRNA Isolation Kit (Qiegene 公司产品） 从总 RNA中分离 poly (A) mRNA。 2ug poly (A) mRNA经逆转录 形成 cDNA。用 Smart cDNA克隆试剂盒（购自 Clontech )将。0^片段定向插入到 pBSK (+) 载体（Clontech公司产品）的多克隆位点上， 转化 DH5 α , 细菌形成 cDNA文库。 用 Dye terminate cycle react ion sequencing kit (Perkin- Elmer公司产品) 禾口 ABI 377 自动测序仪（Perkin-Eimer公司）测定所有克隆的 和 3'末端的序列。 将测定的 cDNA 序列与已有的公共 DNA序列数据库 （Genebank) 进行比较， 结果发现其中一个克隆 0062b09的 cDNA序列为新的 DNA。 通过合成一系列引物对该克隆所含的插入 cDNA片 段进行双向测定。 结果表明， 0062b09克隆所含的全长 cDNA为 1462bp (如 Seq IDN0: 1 所示） ， 从第 1007bP至 1258bP有一个 252bP的开放阅读框架 ( 0RF ) ， 编码一个新 的蛋白质 （如 Seq ID NO: 2所示） 。 我们将此克隆命名为 pBS- 0062b09, 编码的蛋 白质命名为人共济失调 -毛细血管扩张症突变蛋白 15。 实施例 2: 用 RT-PCR方法克隆编码人共济失调 -毛细血管扩张症突变蛋白 15的基因 用胎脑细胞总 RNA为模板， 以 oligo- dT为引物进行逆转录反应合成 cDNA,用Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / phenol / chloroform. Poly (A) mRNA was isolated from total RNA using Quik mRNA Isolation Kit (Qiegene). 2ug poly (A) mRNA is reverse transcribed to form cDNA. Use Smart cDNA Cloning Kit (purchased from Clontech). The 0 ^ fragment was inserted into the multiple cloning site of the pBSK (+) vector (Clontech), and transformed into DH5α. The bacteria formed a cDNA library. Dye terminate cycle react ion sequencing kit (Perkin-Elmer) and ABI 377 automatic sequencer (Perkin-Eimer) were used to determine the sequence of all clones and 3 'ends. The determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and it was found that the cDNA sequence of one of the clones, 062b09, was new DNA. A series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions. The results show that the full-length cDNA contained in clone 0062b09 is 1462bp (as shown in Seq IDN0: 1), and there is an open reading frame (0RF) of 252b P from 1007b P to 1258b P , which encodes a new Protein (as shown in Seq ID NO: 2). We named this clone pBS-0062b09 and the encoded protein was named human ataxia-capillary telangiectasia mutant protein 15. Example 2: Cloning of the gene encoding human ataxia-capillary telangiectasia mutant protein 15 by RT-PCR method Using fetal brain cell total RNA as a template and oligo-dT as a primer for reverse transcription reaction to synthesize cDNA.
Qiagene的试剂盒纯化后，用下列引物进行 PCR扩增： After purification of Qiagene's kit, PCR amplification was performed with the following primers:
Primerl: 5，- GAATGTGGTCATGTCATTTCTAGA -3， (SEQ ID NO: 3)  Primerl: 5,-GAATGTGGTCATGTCATTTCTAGA -3, (SEQ ID NO: 3)
Priraer2: 5，- AAAGCCAACATTTATTATATGTAC -3， (SEQ ID NO: 4)  Priraer2: 5,-AAAGCCAACATTTATTATATGTAC -3, (SEQ ID NO: 4)
Primerl为位于 SEQ ID NO: 1的 5，端的第 lbp开始的正向序列；  Primerl is a forward sequence starting at lbp of the 5th end of SEQ ID NO: 1;
Primer2为 SEQ ID NO: 1的中的 3'端反向序列。  Primer2 is the 3 'end reverse sequence in SEQ ID NO: 1.
扩增反应的条件： 在 50 μ 1的反应体积中含有 50誦 ol/L KC1, 10mniol/L Tris - CI, (pH8.5), 1.5mmol/L MgCl2, 200 μ mol/L dNTP, lOpmol引物， 1U的 Taq DNA聚合 酶（Clontech公司产品）。 在 PE9600型 DNA热循环仪（Perkin- Elmer公司）上按下列条 件反应 25个周期： 94°C 30sec; 55°C 30sec; 72°C 2min。 在 RT- PCR时同时设（3 - act in 为阳性对照和模板空白为阴性对照。 扩增产物用 QIAGEN公司的试剂盒纯化， 用 TA 克隆试剂盒连接到 PCR载体上 （Invitrogen公司产品） 。 DNA序列分析结果表明 PCR 产物的 DNA序列与 SEQ ID NO: 1所示的 1- 1462bp完全相同。 实施例 3: Northern 印迹法分析人共济失调 -毛细血管扩张症突变蛋白 15基因的 表达： Amplification conditions: 50 μl of KC1, 10mniol / L Tris-CI, (pH8.5), 1.5mmol / L MgCl 2 , 200 μmol / L dNTP, lOpmol in a reaction volume of 50 μ 1 Primer, 1U Taq DNA polymerase (Clontech). The reaction was performed on a PE9600 DNA thermal cycler (Perkin-Elmer) under the following conditions for 25 cycles: 94 ° C 30sec; 55 ° C 30sec; 72 ° C 2min. During RT-PCR, (3-act in is the positive control and the template blank is the negative control. The amplified product is purified using QIAGEN's kit, and the TA cloning kit is connected to the PCR vector (Invitrogen's product). DNA The sequence analysis results showed that the DNA sequence of the PCR product was exactly the same as that of 1 to 1462bp shown in SEQ ID NO: 1. Example 3: Northern blot analysis of human ataxia-capillary telangiectasia mutant protein 15 gene expression:
用一歩法提取总 RNA [Anal. Biochem 1987, 162, 156-159]„ 该法包括酸性硫 氰酸胍苯酚 -氯仿抽提。 即用 4M异硫氰酸胍- 25mM柠檬酸钠， 0.2M乙酸钠 （ pH4.0 ) 对组织进行匀浆， 加入 1倍体积的苯酚和 1/5体积的氯仿-异戊醇 （49: 1 ) , 混合 后离心。 吸出水相层， 加入异丙醇 （0.8体积） 并将混合物离心得到 RNA沉淀。 将 得到的 RNA沉淀用 70%乙醇洗涤， 干燥并溶于水中。 用 20μ§ RNA, 在含 20mM 3- ( N- 吗啉代） 丙磺酸 （pH7.0) - 5mM乙酸钠 - ImM EDTA- 2.2M甲醛的 1.2%琼脂糖凝胶上进 行电泳。 然后转移至硝酸纤维素膜上。 用 a- 32P dATP通过随机引物法制备 32P-标记 的 DNA探针。 所用的 DNA探针为图 1所示的 PCR扩增的人共济失调 -毛细血管扩张症 突变蛋白 15编码区序列（1007bp至 1258bp)。 将 32P-标记的探针 (约 2 χ 106cPm/ral ) 与转移了 RNA的硝酸纤维素膜在一溶液中于 42°C杂交过夜， 该溶液包含 50%甲酰胺- 25mMKH2P04 ( pH7.4 ) -5 χ SSC-5 χ Denhardt's溶液和 200 μ g/ml鲑精 DNA。 杂交之后， 将滤膜在 1 X SSC- 0.1°/。SDS中于 55°C洗 30min。 然后， 用 Phosphor Imager进行分析和 定量。 实施例 4: 重组人共济失调 -毛细血管扩张症突变蛋白 15的体外表达、 分离和纯化 根据 SEQ ID N0: 1和图 1所示的编码区序列， 设计出一对特异性扩增引物， 序 列如下： Total RNA extraction using a one-step method [Anal. Biochem 1987, 162, 156-159] This method includes acid guanidinium thiocyanate phenol-chloroform extraction. 4M guanidinium isothiocyanate-25mM sodium citrate, 0.2M acetic acid Sodium (pH4.0) was used to homogenize the tissue, 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) were added, and the mixture was centrifuged. The aqueous phase layer was aspirated and isopropyl alcohol (0.8 Volume) and the mixture was centrifuged to obtain an RNA pellet. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water. Using 20 μ§ of RNA in 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7. 0) - ImM EDTA- 2.2M electrophoresed on a 1.2% agarose formaldehyde gel and transferred onto a nitrocellulose membrane using 32 P- labeled DNA prepared a- 32 P dATP by random priming Method - 5 mM sodium acetate. Probe. The DNA probe used was the PCR amplified human ataxia-capillary telangiectasia mutant protein 15 coding region sequence (1007bp to 1258bp) shown in Figure 1. A 32P-labeled probe (about 2 x 10 6 c P m / ral) 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 χ SSC-5 χ Denhardt's solution and 200 μg / ml salmon sperm DNA. After hybridization, the filter was washed in 1 X SSC-0.1 ° /. SDS at 55 ° C 30 min. Phosphor Imager was then used for analysis and quantification. Example 4: In vitro expression, isolation and purification of recombinant human ataxia-telangiectasia mutant protein 15 According to SEQ ID NO: 1 and the coding region sequence shown in FIG. 1, a pair of specific amplification primers was designed, The sequence is as follows:
Primer 3: 5'- CCCCATATGATGGGAACTGGTTATTTTATTTTC —3， ( Seq ID No: 5 ) Primer 3: 5'- CCCCATATGATGGGAACTGGTTATTTTATTTTC —3, (Seq ID No: 5)
Primer4: 5，一 CCCGAATTCTTATCTTGAAGAGAGAGAAAACTG —3， ( Seq ID No: 6 ) 此两段引物的 5'端分别含有 Ndel和 BamHI酶切位点， 其后分别为目的基因 5'端 和 3 '端的编码序列， Ndel和 BamH I酶切位点相应于表达载体质粒 pET-28 b (+) (No va gen 公司产品， Cat. No.69865.3)上的选择性内切酶位点。 以含有全长目的基因的 pBS- 0062b09质粒为模板， 进行 PCR反应。 PCR反应条件为： 总体积 50 μ 1中含 pBS- 0062b09 质粒 10pg、 弓 I物 Primer- 3和 Primer- 4分另1 J为 lOpmol、 Advantage polymerase Mix ( Clontech公司产品） 1 μ 1。 循环参数： 94。C 20s, 60°C 30s, 68。C 2 min，共 25个 循环。 用 Ndel和 BamHI分别对扩增产物和质粒 pET-28(+)进行双酶切，分别回收大片 段，并用 T4连接酶连接。 连接产物转化用氯化钙法大肠杆细菌 DH5CC,在含卡那霉素 (终浓度 30 g/ml ) 的 LB平板培养过夜后， 用菌落 PCR方法筛选阳性克隆， 并进行 测序。 挑选序列正确的阳性克隆 （pET- 0062b09) 用氯化钙法将重组质粒转化大肠 杆菌 BL21(DE3)PlySs(Novagen公司产品）。 在含卡那霉素 （终浓度 30μ§/ηι1 ) 的 LB 液体培养基中， 宿主菌 BL21 (pET- 0062b09) 在 37°C培养至对数生长期， 加入 IPTG 至终浓度 1讓 ol/L， 继续培养 5小时。 离心收集菌体， 经超声波破菌，离心收集上清， 用能与 6个组氨酸 （ 6His-Tag ) 结合的亲和层析柱 His. Bind Quick Cartridge (Novagen公司产品） 进行层析， 得到了纯化的目的蛋白人共济失调 -毛细血管扩 张症突变蛋白 15。 经 SDS- PAGE电泳， 在 15KDa处得到一单一的条带 （图 2 ) 。 将该 条带转移至 PVDF膜上用 Edams水解法进行 N-端氨基酸序列分析， 结果 N-端 15个氨基 酸与 SEQ ID NO: 2所示的 N-端 15个氨基酸残基完全相同。 实施例 5 抗人共济失调 -毛细血管扩张症突变蛋白 15抗体的产生 Primer4: 5, a CCCGAATTCTTATCTTGAAGAGAGAGAAAACTG-3, (Seq ID No: 6) The 5 'ends of these two primers contain Ndel and BamHI digestion sites, respectively, followed by the coding sequences of the 5' and 3 'ends of the target gene, respectively. The Ndel and BamH I restriction sites correspond to the selective endonuclease sites on the expression vector plasmid pET-28 b (+) (Novagen product, Cat. No. 69865.3). The PCR reaction was performed using the pBS-0062b09 plasmid containing the full-length target gene as a template. The PCR reaction conditions are as follows: a total volume of 50 μl contains 10 pg of pBS- 0062b09 plasmid, Primer-3 and Primer-4, and 1 J is lOpmol, Advantage polymerase Mix (Clontech) 1 μ1. Cycle parameters: 94. C 20s, 60 ° C 30s, 68. C 2 min, a total of 25 cycles. Ndel and BamHI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into the colibacillus DH5CC using the calcium chloride method. After being cultured overnight on an LB plate containing kanamycin (final concentration 30 g / ml), positive clones were selected by colony PCR method and sequenced. Selected positive clones with the correct sequence (pET- 0062b09) the recombinant plasmid by the calcium chloride method to transform E. coli BL21 (DE3) P lySs (Novagen Co.). In LB liquid medium containing kanamycin (final concentration 30μ § / ηι1), the host bacteria BL21 (pET- 0062b09) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 to make ol / L. Continue to cultivate for 5 hours. The bacteria were collected by centrifugation, and the supernatant was collected by centrifugation. The supernatant was collected by centrifugation. The affinity chromatography column His. Bind Quick Cartridge (product of Novagen) was used to obtain 6 histidines (6His-Tag). Purified human ataxia-capillary telangiectasia mutant protein 15. After SDS-PAGE electrophoresis, a single band was obtained at 15 KDa (Figure 2). The band was transferred to a PVDF membrane and the N-terminal amino acid sequence was analyzed by the Edams hydrolysis method. As a result, the 15 amino acids at the N-terminus were identical to the 15 amino acid residues at the N-terminus shown in SEQ ID NO: 2. Example 5 Production of Anti-Ataxia-Capillary Dilatation Mutant 15 Antibody
用多肽合成仪 （PE公司产品） 合成下述人共济失调 -毛细血管扩张症突变蛋 白 15特异性的多肽：  Polypeptide synthesizer (product of PE company) was used to synthesize the following human ataxia-telangiectasia mutant protein 15 specific peptides:
而 2- Me t- G 1 y-Thr-G 1 y- Tyr- Phe- 11 e- Phe- Phe- Phe- G 1 y- G 1 u- G 1 y-Trp-Arg- C00H (SEQ ID NO: 7)。 将该多肽分别与血蓝蛋白和牛血清白蛋白耦合形成复合， 方法参见： Avrameas, et al. Immunochemistry, 1969; 6: 43。 用 4mg上述血蓝蛋白 多肽复合物加上完全弗氏佐剂免疫家兔， 15天后再用血蓝蛋白多肽复合物加不完 全弗氏佐剂加强免疫一次。 采用经 15 g/ml牛血清白蛋白多肽复合物包被的滴定 板做 ELI SA测定兔血清中抗体的滴度。 用蛋白 A-Sepharose从抗体阳性的家免血清 中分离总 IgG。将多肽结合于溴化氰活化的 Sepharose4B柱上，用亲和层析法从总 I gG 中分离抗多肽抗体。 免疫沉淀法证明纯化的抗体可特异性地与人共济失调 -毛细 血管扩张症突变蛋白 15结合。 实施例 6: 本发明的多核苷酸片段用作杂交探针的应用 Whereas 2- Me t- G 1 y-Thr-G 1 y- Tyr- Phe- 11 e- Phe- Phe- Phe- G 1 y- G 1 u- G 1 y-Trp-Arg- C00H (SEQ ID NO : 7). The polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively. For methods, see: Avrameas, et al. Immunochemistry, 1969; 6: 43. Rabbits were immunized with 4 mg of the hemocyanin polypeptide complex plus complete Freund's adjuvant, and 15 days later, the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost immunity once. Titration with 15 g / ml bovine serum albumin peptide complex Plates were made with ELI SA to determine antibody titers in rabbit serum. Protein A-Sepharose was used to isolate total Ig G from antibody-positive home-immunized serum. The peptide was bound to a cyanogen bromide-activated Sepharose4B column, and anti-peptide antibodies were separated from total I gG by affinity chromatography. The immunoprecipitation method proved that the purified antibody could specifically bind to human ataxia-capillary telangiectasia mutant protein 15. Example 6: Application of the polynucleotide fragment of the present invention as a hybridization probe
从本发明的多核苷酸中挑选出合适的寡核苷酸片段用作杂交探针有多方面的 用途， 如用该探针可与不同来源的正常组织或病理组织的基因组或 cDNA文库杂交 以鉴定其是否含有本发明的多核苷酸序列和检出同源的多核苷酸序列，进一歩还可 用该探针检测本发明的多核苷酸序列或其同源的多核苷酸序列在正常组织或病理 组织细胞中的表达是否异常。  Suitable oligonucleotide fragments selected from the polynucleotides of the present invention are used as hybridization probes in a variety of ways. For example, the probes can be used to hybridize to genomic or cDNA libraries of normal tissue or pathological tissue from different sources to It is identified whether it contains the polynucleotide sequence of the present invention and a homologous polynucleotide sequence is detected. Further, the probe can also be used to detect the polynucleotide sequence of the present invention or its homologous polynucleotide sequence in normal tissues or Whether the expression in pathological tissue cells is abnormal.
本实施例的目的是从本发明的多核苷酸 SEQ ID NO: 1 中挑选出合适的寡核苷 酸片段用作杂交探针， 并用滤膜杂交方法鉴定一些组织中是否含有本发明的多核 苷酸序列或其同源的多核苷酸序列。 滤膜杂交方法包括斑点印迹法、 Southern 印 迹法、 Nor thern 印迹法和复印方法等， 它们都是将待测的多核苷酸样品固定在滤 膜上后使用基本相同的歩骤杂交。 这些相同的步骤是： 固定了样品的滤膜首先用 不含探针的杂交缓冲液进行预杂交， 以使滤膜上样品的非特异性的结合部位被载 体和合成的多聚物所饱和。 然后预杂交液被含有标记探针的杂交缓冲液替换， 并 保温使探针与靶核酸杂交。 杂交步骤之后， 未杂交上的探针被一系列洗膜歩骤除 掉。 本实施例利用较高强度的洗膜条件 （如较低盐浓度和较高的温度）， 以使杂交 背景降低且只保留特异性强的信号。 本实施例选用的探针包括两类： 第一类探针 是完全与本发明的多核苷酸 SEQ ID NO: 1相同或互补的寡核苷酸片段； 第二类探 针是部分与本发明的多核苷酸 SEQ ID NO: 1相同或互补的寡核苷酸片段。 本实施 例选用斑点印迹法将样品固定在滤膜上， 在较高强度的的洗膜条件下， 第一类探 针与样品的杂交特异性最强而得以保留。  The purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by a filter hybridization method. Acid sequence or a homologous polynucleotide sequence thereof. Filter hybridization methods include dot blotting, Southern imprinting, Nor thern blotting, and copying methods. They all use the same basic hybridization method after fixing the polynucleotide sample to be tested on the filter. These same steps are as follows: The sample-immobilized filter is first pre-hybridized with a probe-free hybridization buffer to saturate the non-specific binding site of the sample on the filter with the carrier and the synthesized polymer. The pre-hybridization solution is then replaced with a hybridization buffer containing labeled probes and incubated to hybridize the probes to the target nucleic acid. After the hybridization step, unhybridized probes are removed by a series of membrane washes. This embodiment uses 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. In this example, the dot blot method is used to fix the sample on the filter membrane. Under the high-intensity washing conditions, the first type of probe and the sample have the strongest hybridization specificity and are retained.
一、 探针的选用 First, the selection of the probe
从本发明的多核苷酸 SEQ ID NO: 1 中选择寡核苷酸片段用作杂交探针， 应遵 循以下原则和需要考虑的几个方面：  The selection of 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:
1， 探针大小优选范围为 18- 50个核苷酸； 1. The preferred range of probe size is 18-50 nucleotides;
2， GC含量为 30%-70%， 超过则非特异性杂交增加； 2. The GC content is 30% -70%, and the non-specific hybridization increases when it exceeds;
3， 探针内部应无互补区域； 3. There should be no complementary regions inside the probe;
4， 符合以上条件的可作为初选探针， 然后进一步作计算机序列分析， 包括将该 初选探针分别与其来源序列区域 （即 SEQ ID NO: 1 ) 和其它已知的基因组序 列及其互补区进行同源性比较， 若与非靶分子区域的同源性大于 85%或者有超 过 15个连续碱基完全相同， 则该初选探针一般就不应该使用； 4. 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 The column and its complementary region 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 generally
5, 初选探针是否最终选定为有实际应用价值的探针还应进一步由实验确定。 5. Whether the preliminary selection probe is finally selected as a probe with practical application value should be further determined by experiments.
完成以上各方面的分析后挑选并合成以下二个探针：  After completing the above analysis, select and synthesize the following two probes:
探针 1 (probel), 属于第一类探针， 与 SEQ ID NO: 1 的基因片段完全 同源或互补 (41Nt):  Probe 1 (probel), which belongs to the first type of probe, is completely homologous or complementary to the gene fragment of SEQ ID NO: 1 (41Nt):
5'- TGGGAACTGGTTATTTTATTTTCTTCTTTGGTGAGGGATGG -3' ( SEQ ID NO: 8 ) 探针 2 (probe2), 属于第二类探针， 相当于 SEQ ID NO: 1 的基因片段 或其互补片段的替换突变序列 （41Nt):  5'- TGGGAACTGGTTATTTTATTTTCTTCTTTGGTGAGGGATGG -3 '(SEQ ID NO: 8) 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):
5- TGGGAACTGGTTATTTTATTCTCTTCTTTGGTGAGGGATGG -3' ( SEQ ID NO: 9) 献： DNA PROBES G. H. Ke 1 ler; M. M. Manak; Stockton Press, 1989 (USA)以及更常 用的分子克隆实验手册书籍如 《分子克隆实验指南》（ 1998 年第二版） [美]萨姆 布鲁克等著， 科学出版社。  5- TGGGAACTGGTTATTTTATTCTCTTCTTTGGTGAGGGATGG -3 '(SEQ ID NO: 9) Contribution: DNA PROBES GH Ke 1 ler; MM Manak; Stockton Press, 1989 (USA) and more commonly used molecular cloning experiment manual books such as "Molecular Cloning Experiment Guide" (1998 (Second Edition) [US] Sambrook waiting, Science Press.
样品制备：  Sample Preparation:
1, 从新鲜或冰冻组织中提取 DNA  1.Extract DNA from fresh or frozen tissue
歩骤： 1 )将新鲜或新鲜解冻的正常肝组织放入浸在冰上并盛有磷酸盐缓冲液 (PBS) 的平皿中。 用剪刀或手术刀将组织切成小块。 操作中应保持组织湿润。 2) 以 lOOOg离心切碎组织 10分钟。 3 )用冷匀浆缓冲液 ( 0.25mol/L蔗糖； 2 mmol/L Tris-HCl, pH7.5; 25mmol/LnaCl; 25mmol/L MgCl2 ) 悬浮沉淀 （大约 10nil/g )。 4) 在 4nC用电动匀浆器以全速匀浆组织悬液， 直至组织被完全破碎。 5 ) lOOOg 离心 10分钟。 6)用重悬细胞沉淀 （每 0. lg最初组织样品加 l-5ml ), 再以 lOOOg离心 10分钟。 7)用裂解缓冲液重悬沉淀 （每 O. lg最初组织样品加 lml )， 然后接以下 的苯酚抽提法。 Steps: 1) Place fresh or freshly thawed normal liver tissue in a plate immersed in ice and filled with phosphate buffered saline (PBS). Cut the tissue into small pieces with scissors or a scalpel. Keep tissue moist during operation. 2) Centrifuge the tissue at 1,000 g for 10 minutes. 3) Suspend the precipitate (about 10 nil / g) with a cold homogenization buffer (0.25 mol / L sucrose; 2 mmol / L Tris-HCl, pH 7.5; 25 mmol / LnaCl; 25 mmol / L MgCl 2 ). 4) At 4 n C, homogenize the tissue suspension at full speed with an electric homogenizer until the tissue is completely broken. 5) Centrifuge at 1000g for 10 minutes. 6) Resuspend the cell pellet (l-5ml per 0.1 g of the initial tissue sample), and centrifuge at 1,000 g for 10 minutes. 7) Resuspend the pellet with lysis buffer (1 ml per 0.1 g of the initial tissue sample), and then follow the phenol extraction method below.
2, DNA的苯酚抽提法 2, DNA phenol extraction method
歩骤： 1)用 l-10ml 冷 PBS 洗细胞， lOOOg 离心 10分钟。 2)用冷细胞裂解 液重悬浮沉淀的细胞 （lxlO8细胞 /ml ) 最少应用 lOOul 裂解缓冲液。 3)加 SDS 至终浓度为 1%, 如果在重悬细胞之前将 SDS直接加入到细胞沉淀中， 细胞可能会 形成大的团块而难以破碎， 并降低的总产率。 这一点在抽提〉107细胞时特别严重。 4 )加蛋白酶 K至终浓度 200ug/ml。 5 ) 50°C保温反应 1 小时或在 37°C轻轻振摇 过夜。 6)用等体积苯酚： 氯仿： 异戊醇 （ 25: 24: 1 ) 抽提， 在小离心机管中离 心 10分钟。 两相应清楚分离， 否则重新进行离心。 7) 将水相转移至新管。 8)用 等体积氯仿： 异戊醇 （24: 1)抽提， 离心 10分钟。 9)将含 DNA的水相转移至新 管。 然后进行 DNA的纯化和乙醇沉淀。 Steps: 1) Wash the cells with 1-10ml of cold PBS and centrifuge at 1000g for 10 minutes. 2) Resuspend the pelleted cells with cold cell lysate (lx10 8 cells / ml). Use a minimum of 100ul lysis buffer. 3) Add SDS to a final concentration of 1%. If SDS is directly added to the cell pellet before resuspending the cells, the cells may form large clumps that are difficult to break, and reduce the overall yield. This is particularly serious when extracting> 10 7 cells. 4) Add proteinase K to a final concentration of 200ug / ml. 5) Incubate at 50 ° C for 1 hour or shake gently at 37 ° C overnight. 6) Extract with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge in a small centrifuge tube for 10 minutes. The two should be clearly separated, otherwise centrifuge again. 7) Transfer the water phase to a new tube. 8) Extract with an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes. 9) Transfer the DNA-containing aqueous phase to a new Tube. The DNA was then purified and ethanol precipitated.
3, DNA的纯化和乙醇沉淀 3, DNA purification and ethanol precipitation
歩骤： 1)将 1/10体积 2mol/L醋酸钠和 2倍体积冷 100%乙醇加到 DNA溶液 中， 混匀。 在 -20°C放置 1小时或至过夜。 2) 离心 10分钟。 3)小心吸出或倒出 乙醇。 4 )用 70%冷乙醇 500ul 洗涤沉淀， 离心 5分钟。 5 ) 小心吸出或倒出乙醇。 用 500ul冷乙醇洗涤沉淀， 离心 5分钟。 6)小心吸出或倒出乙醇， 然后在吸水纸 上倒置使残余乙醇流尽。 空气干燥 10-15 分钟， 以使表面乙醇挥发。 注意不要使 沉淀完全干燥， 否则较难重新溶解。 7) 以小体积 TE或水重悬 DNA 沉淀。 低速涡 旋振荡或用滴管吹吸， 同时逐渐增加 TE， 混合至 DNA充分溶解， 每 1- 5 x l0ft细胞 所提取的大约加 lul。 Steps: 1) Add 1/10 volume of 2mol / L sodium acetate and 2 volumes of cold 100% ethanol to the DNA solution and mix. Leave at -20 ° C for 1 hour or overnight. 2) Centrifuge for 10 minutes. 3) Carefully aspirate or pour out the ethanol. 4) Wash the pellet with 500ul of 70% cold ethanol and centrifuge for 5 minutes. 5) Carefully aspirate or pour out the ethanol. Wash the pellet with 500ul of cold ethanol and centrifuge for 5 minutes. 6) Carefully aspirate or pour out the ethanol, then invert on the absorbent paper to drain off the residual ethanol. Air dry for 10-15 minutes to allow the surface ethanol to evaporate. Be careful not to allow the pellet to dry completely, otherwise it will be more difficult to re-dissolve. 7) Resuspend the DNA pellet in a small volume of TE or water. Vortex at low speed or suck with a pipette while gradually increasing TE, mix until the DNA is fully lysed, add approximately 1 ul per 1-5 x 10 ft cells.
以下第 8-13步骤仅用于必须除去污染时， 否则可直接进行第 14步骤。  The following steps 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
8 ) 将 RNA酶 A加到 DNA溶液中， 终浓度为 100ug/ml, 37°C保温 30分钟。 9 )加 入 SDS和蛋白酶 K， 终浓度分别为 0.5%和 100ug/ml。 37°C保温 30分钟。 10)用 等体积的苯酚： 氯仿： 异戊醇 （ 25: 24： 1)抽提反应液， 离心 10 分钟。 11 ) 小 心移出水相， 用等体积的氯仿： 异戊醇 （24: 1 ) 重新抽提， 离心 10 分钟。 12 ) 小心移出水相， 加 1/10体积 2mol/L 醋酸钠和 2.5 体积冷乙醇， 混匀置 - 20°C 1 小时。 13) 用 70%乙醇及 100%乙醇洗涤沉淀， 空气干燥， 重悬核酸， 过程同第 3 - 6歩骤。 14) 测定 A26。和 A28。以检测 DNA的纯度及产率。 15 ) 分装后存放于 -20 C。 样膜的制备： 8) Add RNase A to the DNA solution to a final concentration of 100ug / ml, and incubate at 37 ° C for 30 minutes. 9) Add SDS and proteinase K, the final concentrations are 0.5% and 100ug / ml, respectively. Incubate at 37 ° C for 30 minutes. 10) Extract the reaction solution with an equal volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and centrifuge for 10 minutes. 11) Carefully remove the aqueous phase and re-extract with an equal volume of chloroform: isoamyl alcohol (24: 1) and centrifuge for 10 minutes. 12) Carefully remove the water phase, add 1/10 volume of 2mol / L sodium acetate and 2.5 volumes of cold ethanol, and mix well--20 ° C for 1 hour. 13) Wash the pellet with 70% ethanol and 100% ethanol, air dry, and resuspend the nucleic acid. The process is the same as steps 3-6. 14) Measure A 26 . And A 28 . To detect the purity and yield of DNA. 15) Store at -20 C after packaging. Preparation of sample film:
1) 取 4 x 2 张适当大小的硝酸纤维素膜 （NC 膜）， 用铅笔在其上轻轻标出点样 位置及样号， 每一探针需两张 NC膜， 以便在后面的实验步骤中分别用高强度条件 和强度条件洗膜 。  1) Take 4 x 2 pieces of nitrocellulose membranes (NC membranes) of appropriate size, and mark the spotting position and sample number lightly with a pencil. Two NC membranes are required for each probe for subsequent experiments. In the step, the film is washed with high-strength conditions and strength conditions, respectively.
2) 吸取及对照各 15微升， 点于样膜上， 在室温中晾干。  2) Pipette and control 15 microliters each, spot on the sample film, and dry at room temperature.
3 ) 置于浸润有 0. Imol/LNaOH, 1.5mol/LNaCl的滤纸上 5分钟 (两次）， 晾干置 于浸润有 0.5mol/L Tris-HCl (pH7.0 )， 3raol/LNaCl的滤纸上 5分钟 (两次）, 晾 干。  3) Place on filter paper impregnated with 0.1 mol / L NaOH, 1.5 mol / L NaCl for 5 minutes (twice), dry and place on filter paper impregnated with 0.5 mol / L Tris-HCl (pH 7.0), 3 raol / L NaCl Allow to dry for 5 minutes (twice).
4) 夹于干净滤纸中， 以铝箔包好， 60- 80°C真空干燥 2小时。  4) Caught in clean filter paper, wrapped in aluminum foil, and dried under vacuum at 60-80 ° C for 2 hours.
探针的标记  Labeling of probes
1 ) 3 μ lProbe ( 0.10D/10 μ 1 )，加入 2 μ IKinase缓冲液， 8-10 uCi γ- 32P- dATP+2U Kinase, 以补加至终体积 20 μ 1。 1) 3 μl Probe (0.10D / 10 μ 1), add 2 μ IKinase buffer, 8-10 uCi γ- 32 P- dATP + 2U Kinase, to make up to a final volume of 20 μ 1.
2 ) 37 °C 保温 2小时。  2) Incubate at 37 ° C for 2 hours.
3)加 1/5体积的溴酚蓝指示剂 ( BPB  3) Add 1/5 volume of bromophenol blue indicator (BPB
4 ) 过 Sephadex G-50柱。 5 ) 至有 32P- Probe洗出前开始收集第一峰（可用 Monitor监测）。 4) Pass through a Sephadex G-50 column. 5) Before the 32 P-Probe is washed out, start collecting the first peak (can be monitored by Monitor).
6 ) 5滴 /管， 收集 10-15管。  6) 5 drops / tube, collect 10-15 tubes.
7 )用液体闪烁仪监测同位素量  7) Monitor the amount of isotope with a liquid scintillator
8 ) 合并第一峰的收集液后即为所需制备的 32P-Probe (第二峰为游离 γ-32Ρ- dATP )。 8) The combined solution after the first peak was collected as 32 P-Probe (second peak to prepare the desired free γ- 32 Ρ- dATP).
预杂交  Pre-hybridization
将样膜置于塑料袋中，加入 3- 10mg预杂交液（ 10xDenhardfs;6xSSC, 0.1 mg /ml CT DNA (小牛胸腺 DM)。）， 封好袋口后， 68°C水浴摇 2小时。  Put the sample film in a plastic bag, add 3-10 mg of prehybridization solution (10xDenhardfs; 6xSSC, 0.1 mg / ml CT DNA (calf thymus DM).), Seal the bag, and shake at 68 ° C for 2 hours in a water bath.
杂交  Cross
将塑料袋剪去一角， 加入制备好的探针， 封好袋口后， 42°C水浴摇过夜。 洗膜：  Cut a corner of the plastic bag, add the prepared probe, seal the bag, and shake it at 42 ° C in a water bath overnight. Wash film:
高强度洗膜：  High-intensity washing film:
1 ) 取出已杂交好的样膜。  1) Take out the hybridized sample membrane.
2 ) 2xSSC, 0.1%SDS中， 40°C洗 15分钟 （ 2次）。  2) 2xSSC, 0.1% SDS, wash at 40 ° C for 15 minutes (twice).
3 ) 0. lxSSC, 0.1%SDS中， 40。C洗 15分钟 （ 2次）。  3) 0.1xSSC, 0.1% SDS, 40. C Wash for 15 minutes (twice).
4 ) 0. lxSSC, 0.1%SDS中， 55°C洗 30分钟 （2次）， 室温晾干。 低强度洗膜：  4) In 0.1xSSC, 0.1% SDS, wash at 55 ° C for 30 minutes (twice), and dry at room temperature. Low-intensity washing film:
1 ) 取出已杂交好的样膜。  1) Take out the hybridized sample membrane.
2 ) 2xSSC, 0.1%SDS中， 37°C洗 15分钟 （ 2次）。  2) 2xSSC, 0.1% SDS, wash at 37 ° C for 15 minutes (twice).
3 ) 0. lxSSC, 0.1%SDS中， 37°C洗 15分钟 （ 2次）。  3) 0.1xSSC, 0.1% SDS, wash at 37 ° C for 15 minutes (twice).
4 ) 0. lxSSC, 0.1%SDS中, 40°C洗 15分钟 （2次）, 室温晾干。  4) 0.1xSSC, 0.1% SDS, wash at 40 ° C for 15 minutes (twice), and dry at room temperature.
X-光自显影： X-ray auto-development:
-70°C, X-光自显影 （压片时间根据杂交斑放射性强弱而定）。  -70 ° C, X-ray autoradiography (pressing time depends on the radioactivity of the hybrid spot).
实验结果：  Experimental results:
采用低强度洗膜条件所进行的杂交实验， 以上两个探针杂交斑放射性强弱没 有明显区别； 而釆用高强度洗膜条件所进行的杂交实验， 探针 1 的杂交斑放射性 强度明显强于另一个探针杂交斑的放射性强度。 因而可用探针 1 定性和定量地分 析本发明的多核苷酸在不同组织中的存在和差异表达。  The hybridization experiments performed under low-intensity membrane washing conditions showed no significant difference in the radioactive intensity of the above two probes. However, in the hybridization experiments performed under high-intensity membrane washing conditions, the radioactive intensity of probe 1 was significantly stronger. To the radioactive intensity of the hybridization spot of another probe. Therefore, the presence and differential expression of the polynucleotide of the present invention in different tissues can be analyzed qualitatively and quantitatively with the probe 1.
实施例 7 DNA Microarray 基因芯片或基因微矩阵 （DNA Microarray) 是目前许多国家实验室和大制药 公司都在着手研制和开发的新技术， 它是指将大量的靶基因片段有序地、 高密度 地排列在玻璃、 硅等载体上， 然后用荧光检测和计算机软件进行数据的比较和分 析， 以达到快速、 高效、 高通量地分析生物信息的目的。 本发明的多核苷酸可作 为靶 DNA 用于基因芯片技术用于高通量研究新基因功能； 寻找和筛选组织特异性 新基因特别是肿瘤等疾病相关新基因； 疾病的诊断， 如遗传性疾病。 其具体方法 步骤在文献中已有多种报道， 如可参阅文献 DeRisi， J. L. , Lyer, V. &Brown, P.0. (1997) Science278, 680-686.及文献 Helle, R. A. , Schema, M. , Chai, A.， Shalom, D.， (1997) PNAS 94: 2150-2155. Example 7 DNA Microarray Gene microarrays or DNA microarrays are new technologies currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information. The polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research of new gene functions; search for and screen new tissue-specific genes, especially new genes related to diseases such as tumors; diagnosis of diseases such as hereditary diseases . The specific method steps have been reported in the literature. For example, see DeRisi, JL, Lyer, V. & Brown, P.0. (1997) Science 278, 680-686. And Helle, RA, Schema, M. , Chai, A., Shalom, D., (1997) PNAS 94: 2150-2155.
(一） 点样  (A) spotting
备种不同的全长 cDNA共计 4000条多核苷酸序列作为靶 DM,其中包括本发明 的多核苷酸。 将它们分别通过 PCR 进行扩增， 纯化所得扩增产物后将其浓度调到 500ng/ul 左右， 用 Cartesian 7500 点样仪（购自美国 Cartesian公司）点于玻璃 介质上， 点与点之间的距离为 280 μη!。 将点样后的玻片进行水合、 干燥、 置于紫 外交联仪中交联， 洗脱后干燥使 DNA 固定在玻璃片上制备成芯片。 其具体方法歩 骤在文献中已有多种报道， 本实施例的点样后处理步骤是：  A total of 4,000 polynucleotide sequences of different full-length cDNAs were prepared as the target DM, including the polynucleotide of the present invention. They were amplified by PCR respectively. After purification, the amplified product was adjusted to a concentration of about 500 ng / ul, and spotted on a glass medium using a Cartesian 7500 spotter (purchased from Cartesian, USA). The distance is 280 μη !. The spotted slides were hydrated, dried, and cross-linked in a purple diplomatic coupling instrument. After elution, the DNA was fixed on a glass slide to prepare a chip. The specific method steps have been variously reported in the literature, and the post-spotting processing steps of this embodiment are:
1. 潮湿环境中水合 4小时；  1. Hydration in a humid environment for 4 hours;
2. 0.2%SDS洗涤 1分钟；  2. 0.2% SDS was washed for 1 minute;
3. ddH20洗涤两次， 每次 1分钟； 3. Wash twice with ddH 2 0 for 1 minute each time;
4. NaBH4封闭 5分钟； 4. NaBH 4 is blocked for 5 minutes;
5. 95°C水中 2分钟；  5. 95 ° C water for 2 minutes;
6. 0.2%SDS洗涤 1分钟；  6. Wash with 0.2% SDS for 1 minute;
7. ddH20冲洗两次； 7. Rinse twice with ddH 2 0;
8. 凉干， 25°C储存于暗处备用。  8. Dry and store at 25 ° C in the dark for future use.
(二）探针标记  (Two) probe marking
用一步法分别从人体混合组织与机体特定组织 （或经过刺激的细胞株） 中抽 提总 mRNA, 并用 Oligotex mRNA Midi Kit (购自 QiaGen公司）纯化 mRNA，通过反 转录分别将荧光试剂 Cy3dUTP(5- Amino- propargy卜 2'- deoxyuridine 5'- triphate cou led to Cy3 fluorescent dye, 购自 Amersham Phamacia Biotech公司)标记 人体混合组织的 mRNA,用荧光试剂 Cy5dUTP (5- Amino- propargyl- 2'- deoxyur idine 5'-tr iphate coupled to Cy5 fluorescent dye, 购自 Amersham Phamacia Biotech 公司）标记机体特定组织 （或经过刺激的细胞株） mRNA, 经纯化后制备出探针。 具 体歩骤参照及方法见： Schena, Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and mRNA was purified with Oligotex mRNA Midi Kit (purchased from QiaGen). The fluorescent reagent Cy3dUTP ( 5- Amino- propargy 2'- deoxyuridine 5'- triphate cou led to Cy3 fluorescent dye, purchased from Amersham Phamacia Biotech) was used to label mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5- Amino- propargyl- 2'- deoxyur idine 5'-tr iphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech company, labeled the body's specific tissue (or stimulated cell line) mRNA, and purified the probe to prepare a probe. For specific steps and methods, see: Schena,
M.， Shalon, D. , Heller, R. (1996) Proc. Natl. Acad. Sci. USA. Vol.93: 10614- 10619. Schena, M. , Shalon, Dar i. , Davis, R. W. (1995) Science.270. (20): 467-480. M., Shalon, D., Heller, R. (1996) Proc. Natl. Acad. Sci. USA. Vol. 93: 10614- 10619. Schena, M., Shalon, Dar i., Davis, RW (1995) Science.270. (20): 467-480.
(三） 杂交 (Three) cross
分别将来自以上两种组织的探针与芯片一起在 UniHyb™ Hybridization Combine the probes from the two tissues with the chips in UniHyb ™ Hybridization
Solution (购自 TeleChem公司）杂交液中进行杂交 16 小时， 室温用洗涤液 （ 1 x SSC, 0.2%SDS ) 洗涤后用 ScanArray 3000扫描仪 (购自美国 General Scanning公 司）进行扫描， 扫描的图象用 Imagene软件 （美国 Biodiscovery公司） 进行数据 分析处理， 算出每个点的 Cy3/Cy5比值。 Solution (purchased from TeleChem) was used for 16 hours of hybridization, and washed with a washing solution (1 x SSC, 0.2% SDS) at room temperature, and then scanned with a ScanArray 3000 scanner (purchased from General Scanning, USA). The scanned image Imagene software (Biodiscovery, USA) was used for data analysis and processing to calculate the Cy3 / Cy5 ratio of each point.
以上机体特定组织 （或经过刺激的细胞株）分别为胸腺、 睾丸、 肌肉、 脾脏、 肺、 皮肤、 甲状腺、 肝、 PMA+的 Ecv304细胞株、 PMA-的 Ecv304细胞株、 未饥饿的 L02细胞株、 砷刺激 1小时的 L02细胞株以及前列腺组织。 根据这 13个 Cy3/Cy5比值 绘出折方图。 （图 1 ) 。 由图可见本发明所述的人共济失调 -毛细血管扩张症突变 蛋白 15和人共济失调 -毛细血管扩张症突变基因的蛋白产物表达谱很相似。 工业实用性  The above specific tissues (or stimulated cell lines) are thymus, testis, muscle, spleen, lung, skin, thyroid, liver, PMA + Ecv304 cell line, PMA- Ecv304 cell line, non-starved L02 cell line, Arsenic stimulated the L02 cell line and prostate tissue for 1 hour. Draw a graph based on these 13 Cy3 / Cy5 ratios. (figure 1 ) . It can be seen from the figure that the expression profile of the protein product of the human ataxia-telangiectasia mutant protein 15 and the human ataxia-telangiectasia mutant gene according to the present invention are very similar. Industrial applicability
本发明的多肽以及该多肽的拮抗剂、 激动剂和抑制剂可直接用于疾病治疗， 例如， 可治疗恶性肿瘤、 肾上腺缺乏症、 皮肤病、 各类炎症、 HIV 感染和免疫 性疾病等。  The polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat malignant tumors, adrenal deficiency, skin diseases, various inflammations, HIV infections and immune diseases.
共济失调 -毛细血管扩张症是一种常染色体隐性遗传病， 其症状是小脑共 济失调， 毛细血管扩张， 免疫系统缺陷、 易患恶性肿瘤 （特别是白血病和淋巴 瘤） 等。 共济失调 -毛细血管扩张症是由于共济失调 -毛细血管扩张症突变基 因 （ATM, mutated in A-T) 突变而引起的。 ATM基因产物在细胞周期的调节中 起作用， 另外还在 DNA 损伤的反应中起一定作用。 A-T 的杂合子易患肿瘤， 特 别是女性的乳腺癌。  Ataxia-Capillary telangiectasia is an autosomal recessive disorder with symptoms such as ataxia of the cerebellum, telangiectasia, defective immune system, and susceptibility to malignant tumors (especially leukemia and lymphoma). Ataxia-Capillary vasodilation is caused by ataxia-Capillary telangiectasia mutation (ATM, mutated in A-T) mutation. ATM gene products play a role in the regulation of the cell cycle and also play a role in the response to DNA damage. A-T heterozygotes are susceptible to tumors, especially breast cancer in women.
本发明的多肽的表达谱与人共济失调 -毛细血管扩张症突变基因的蛋白产 物的表达谱相一致， 两者具有相似的生物学功能。 它在体内有着各种重要的作 用， 主要调节着体内免疫系统、 细胞增殖、 小脑、 毛细血管等多种组织器官的 生理功能， 其表达异常将在上述组织细胞产生异常， 并产生相关的疾病。  The expression profile of the polypeptide of the present invention is consistent with the expression profile of the protein product of the human ataxia-telangiectasia mutant gene, and both have similar biological functions. It has a variety of important functions in the body, mainly regulating the physiological functions of various tissues and organs in the body such as the immune system, cell proliferation, cerebellum, capillaries, etc. Abnormal expression will cause abnormalities in the above-mentioned tissue cells and cause related diseases.
由此可见， 本发明的人共济失调 -毛细血管扩张症突变蛋白 15 的表达异常 将产生各种疾病尤其是共济失调 -毛细血管扩张症、 乳腺癌、 各种肿瘤、 胚胎 发育紊乱症、 生长发育障碍性疾病、 免疫性疾病、 炎症， 这些疾病包括但不限 于： 各种组织的肿瘤： 乳腺癌， 胃癌， 肝癌， 肺癌， 食管癌， 白血病， 淋巴瘤， 甲状腺肿瘤， 子宫肌瘤， 神经细胞瘤， 星形细胞瘤， 室管膜瘤， 胶质细胞瘤， 神经纤维瘤， 结肠癌， 黑色素瘤， 膀胱癌， 子宫癌， 子宫内膜癌， 结肠癌， 胸 腺肿瘤， 鼻咽癌， 喉癌， 气管肿瘤， 纤维瘤， 纤维肉瘤， 脂肪瘤， 脂肪肉瘤 胚胎发育紊乱症： 先天性流产， 腭裂， 肢体缺如， 肢体分化障碍， 房间隔 缺损， 神经管缺陷， 先天性脑积水， 先天性青光眼或白内障， 先天性耳聋 It can be seen that the abnormal expression of the human ataxia-capillary telangiectasia mutant protein 15 will produce various diseases, especially ataxia-capillary telangiectasia, breast cancer, various tumors, embryonic developmental disorders, Growth disorders, immune diseases, inflammation, these diseases include but are not limited to: Tumors of various tissues: breast cancer, stomach cancer, liver cancer, lung cancer, esophageal cancer, leukemia, lymphoma, thyroid tumors, 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, tracheal tumor, fibroid, fibrosarcoma, lipoma, liposarcoma, embryonic developmental disorders Symptoms: Congenital abortion, cleft palate, limb loss, limb differentiation disorder, atrial septal defect, neural tube defect, congenital hydrocephalus, congenital glaucoma or cataract, congenital deafness
生长发育障碍性疾病： 精神发育迟缓， 脑发育障碍， 皮肤、 脂肪和肌肉发 育不良性疾病， 骨与关节发育不良性疾病， 各种代谢缺陷病， 呆小症， 侏儒症， 库兴综合这征， 性发育迟缓症  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
免疫性疾病： 普通易变免疫缺陷病， 原发性 B 淋巴细胞免疫缺陷病， 原发 性 T 淋巴细胞免疫缺陷病， 获得性免疫缺陷综合症， 系统性红斑狼疮， 类风湿 性关节炎，  Immune diseases: common variable immunodeficiency disease, primary B lymphocyte immunodeficiency disease, primary T lymphocyte immunodeficiency disease, acquired immunodeficiency syndrome, systemic lupus erythematosus, rheumatoid arthritis,
炎症： 慢性活动性肝炎， 结节病， 多肌炎， 慢性鼻炎， 慢性胃炎， 脑脊髓 多发性硬化， 肾小球性肾炎， 心肌炎， 心肌病， 动脉粥样硬化， 胃溃疡， 子宫 颈炎， 各种感染性炎症  Inflammation: chronic active hepatitis, sarcoidosis, polymyositis, chronic rhinitis, chronic gastritis, cerebrospinal multiple sclerosis, glomerulonephritis, myocarditis, cardiomyopathy, atherosclerosis, gastric ulcer, cervicitis, Various infectious inflammations
本发明的人共济失调 -毛细血管扩张症突变蛋白 15 的表达异常还将产生某 些遗传性， 血液性疾病等。  The abnormal expression of the human ataxia-telangiectasia mutant protein 15 of the present invention will also produce certain hereditary, hematological diseases and the like.
本发明的多肽以及该多肽的拮抗剂、 激动剂和抑制剂可直接用于疾病治疗， 例如， 可治疗各种疾病尤其是共济失调 -毛细血管扩张症、 乳腺癌、 各种肿瘤、 胚胎发育紊乱症、 生长发育障碍性疾病、 免疫性疾病、 炎症， 某些遗传性， 血 液性疾病等。 本发明也提供了筛选化合物以鉴定提高（激动剂）或阻遏（拮抗剂）人共济失 调 -毛细血管扩张症突变蛋白 15 的药剂的方法。 激动剂提高人共济失调 -毛细 血管扩张症突变蛋白 15刺激细胞增殖等生物功能， 而拮抗剂阻止和治疗与细胞 过度增殖有关的紊乱如各种癌症。 例如， 能在药物的存在下， 将哺乳动物细胞 或表达人共济失调 -毛细血管扩张症突变蛋白 15 的膜制剂与标记的人共济失调 -毛细血管扩张症突变蛋白 15 —起培养。 然后测定药物提高或阻遏此相互作用 的能力。  The polypeptide of the present invention and the antagonists, agonists and inhibitors of the polypeptide can be directly used in the treatment of diseases, for example, it can treat various diseases, especially ataxia-capillary vasodilation, breast cancer, various tumors, and embryo development. Disorders, growth disorders, immune disorders, inflammation, certain hereditary, hematological diseases, etc. The invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) human ataxia-capillary telangiectasia mutant protein 15. Agonists enhance human ataxia-capillary vasodilator mutant protein 15 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to cell proliferation, such as various cancers. For example, a mammalian cell or a membrane preparation expressing human ataxia-capillary telangiectasia mutant protein 15 can be cultured with a labeled human ataxia-capillary telangiectasia mutant protein 15 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
人共济失调 -毛细血管扩张症突变蛋白 15的拮抗剂包括筛选出的抗体、 化 合物、 受体缺失物和类似物等。 人共济失调 -毛细血管扩张症突变蛋白 1 5 的拮 抗剂可以与人共济失调 -毛细血管扩张症突变蛋白 1 5结合并消除其功能， 或是 抑制该多肽的产生， 或是与该多肽的活性位点结合使该多肽不能发挥生物学功 能。 Antagonists of human ataxia-telangiectasia mutant protein 15 include antibodies, compounds, receptor deletions, and the like that have been screened. Antagonist of human ataxia-capillary telangiectasia mutant protein 15 can bind to human ataxia-capillary telangiectasia mutant protein 15 and eliminate its function, or inhibit the production of the polypeptide, or with the polypeptide Binding of the active site prevents the polypeptide from performing biological functions can.
在筛选作为拮抗剂的化合物时， 可以将人共济失调 -毛细血管扩张症突变 蛋白 15加入生物分析测定中， 通过测定化合物对人共济失调 -毛细血管扩张症 突变蛋白 15 和其受体之间相互作用的影响来确定化合物是否是拮抗剂。 用上述 筛选化合物的同样方法， 可以筛选出起拮抗剂作用的受体缺失物和类似物。 能 与人共济失调 -毛细血管扩张症突变蛋白 15结合的多肽分子可通过筛选由各种 可能组合的氨基酸结合于固相物组成的随机多肽库而获得。 筛选时， 一般应对 人共济失调 -毛细血管扩张症突变蛋白 15分子进行标记。  When screening compounds as antagonists, human ataxia-capillary vasodilatation mutant protein 15 can be added to the bioanalytical assay. The effect of this interaction is used to determine whether the 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 ataxia-capillary telangiectasia mutant protein 15 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. In screening, 15 molecules of ataxia-telangiectasia mutant protein should generally be labeled.
本发明提供了用多肽， 及其片段、 衍生物、 类似物或它们的细胞作为抗原 以生产抗体的方法。 这些抗体可以是多克隆抗体或单克隆抗体。 本发明还提供 了针对人共济失调 -毛细血管扩张症突变蛋白 15抗原决定簇的抗体。 这些抗体 包括（但不限于）： 多克隆抗体、 单克隆抗体、 嵌合抗体、 单链抗体、 Fab 片段 和 Fab表达文库产生的片段。  The present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies. The invention also provides antibodies directed against the human ataxia-capillary telangiectasia mutant protein 15 epitope. These antibodies include (but are not limited to): polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
多克隆抗体的生产可用人共济失调 -毛细血管扩张症突变蛋白 15直接注射 免疫动物 （如家兔， 小鼠， 大鼠等） 的方法得到， 多种佐剂可用于增强免疫反 应， 包括但不限于弗氏佐剂等。 制备人共济失调 -毛细血管扩张症突变蛋白 15 的单克隆抗体的技术包括但不限于杂交瘤技术 （Kohler and Milstein. Nature, 1975, 256: 495-497) , 三瘤技术， 人 Β-细胞杂交瘤技术， EBV-杂交瘤 技术等。 将人恒定区和非人源的可变区结合的嵌合抗体可用已有的技术生产 (Morrison et al，PNAS, 1985， 81: 6851)。而已有的生产单链抗体的技术（U. S. Pat No.4946778)也可用于生产抗人共济失调 -毛细血管扩张症突变蛋白 15 的单链 抗体。  Polyclonal antibodies can be produced by direct injection of human ataxia-capillary telangiectasia mutant protein 15 into immunized animals (such as rabbits, mice, rats, etc.). A variety of adjuvants can be used to enhance the immune response, including but It is not limited to Freund's adjuvant and the like. Techniques for preparing monoclonal antibodies to human ataxia-telangiectasia mutant protein 15 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta cells Hybridoma technology, EBV-hybridoma technology, etc. Chimeric antibodies that bind human constant regions and non-human-derived variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). The existing technology for producing single-chain antibodies (U.S. Pat No. 4946778) can also be used to produce single-chain antibodies against human ataxia-capillary telangiectasia mutant protein 15.
抗人共济失调 -毛细血管扩张症突变蛋白 15的抗体可用于免疫组织化学技 术中， 检测活检标本中的人共济失调 -毛细血管扩张症突变蛋白 15。  Anti-human ataxia-telangiectasia mutant protein 15 antibody can be used in immunohistochemical techniques to detect human ataxia-telangiectasia mutant protein 15 in biopsy specimens.
与人共济失调 -毛细血管扩张症突变蛋白 15结合的单克隆抗体也可用放射 性同位素标记， 注入体内可跟踪其位置和分布。 这种放射性标记的抗体可作为 一种非创伤性诊断方法用于肿瘤细胞的定位和判断是否有转移。  Monoclonal antibodies that bind to human ataxia-telangiectasia mutant protein 15 can also be labeled with radioactive isotopes 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.
抗体还可用于设计针对体内某一特殊部位的免疫毒素。 如人共济失调 -毛 细血管扩张症突变蛋白 15 高亲和性的单克隆抗体可与细菌或植物毒素（如白喉 毒素， 蓖麻蛋白， 红豆碱等）共价结合。 一种通常的方法是用巯基交联剂如 SPDP, 攻击抗体的氨基， 通过二硫键的交换， 将毒素结合于抗体上， 这种杂交抗体可 用于杀灭人共济失调 -毛细血管扩张症突变蛋白 15阳性的细胞。  Antibodies can also be used to design immunotoxins that target a particular part of the body. Such as human ataxia-capillary telangiectasia mutant protein 15 High-affinity monoclonal antibodies can covalently bind to bacterial or phytotoxins (such as diphtheria toxin, ricin, ormosine, etc.). A common method is to attack the amino group of an antibody with a thiol crosslinker 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 ataxia-capillary vasodilation Mutein 15 positive cells.
本发明中的抗体可用于治疗或预防与人共济失调 -毛细血管扩张症突变蛋 白 15相关的疾病。 给予适当剂量的抗体可以刺激或阻断人共济失调 -毛细血管 扩张症突变蛋白 15的产生或活性。 The antibodies in the present invention can be used to treat or prevent ataxia-capillary telangiectasia mutant eggs White 15 related diseases. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human ataxia-telangiectasia mutant protein 15.
本发明还涉及定量和定位检测人共济失调 -毛细血管扩张症突变蛋白 1 5水 平的诊断试验方法。 这些试验是本领域所熟知的， 且包括 FI SH测定和放射免疫 测定。 试验中所检测的人共济失调 -毛细血管扩张症突变蛋白 15水平， 可以用 作解释人共济失调 -毛细血管扩张症突变蛋白 15在各种疾病中的重要性和用于 诊断人共济失调 -毛细血管扩张症突变蛋白 1 5起作用的疾病。  The present invention also relates to a diagnostic test method for quantitatively and locally detecting human ataxia-capillary vasodilation mutant protein level 15. These tests are well known in the art and include FI SH assays and radioimmunoassays. Human ataxia-capillary telangiectasia mutant protein 15 levels detected in the test can be used to explain the importance of human ataxia-capillary telangiectasia mutant protein 15 in various diseases and to diagnose human ataxia Dysregulation-A disease in which telangiectasia mutant protein 15 functions.
本发明的多肽还可用作肽谱分析， 例如， 多肽可用物理的、 化学或酶进行 特异性切割， 并进行一维或二维或三维的凝胶电泳分析，更好的是进行质谱分 析。  The polypeptide of the present invention can also be used for peptide mapping analysis. For example, 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.
编码人共济失调 -毛细血管扩张症突变蛋白 1 5的多核苷酸也可用于多种治 疗目的。 基因治疗技术可用于治疗由于人共济失调 -毛细血管扩张症突变蛋白 15 的无表达或异常 /无活性表达所致的细胞增殖、 发育或代谢异常。 重组的基 因治疗载体（如病毒载体）可设计用于表达变异的人共济失调 -毛细血管扩张症 突变蛋白 1 5， 以抑制内源性的人共济失调 -毛细血管扩张症突变蛋白 1 5 活性。 例如， 一种变异的人共济失调 -毛细血管扩张症突变蛋白 15 可以是缩短的、 缺 失了信号传导功能域的人共济失调 -毛细血管扩张症突变蛋白 15， 虽可与下游 的底物结合， 但缺乏信号传导活性。 因此重组的基因治疗载体可用于治疗人共 济失调 -毛细血管扩张症突变蛋白 15表达或活性异常所致的疾病。 来源于病毒 的表达载体如逆转录病毒、 腺病毒、 腺病毒相关病毒、 单纯疱疹病毒、 细小病 毒等可用于将编码人共济失调 -毛细血管扩张症突变蛋白 15 的多核苷酸转移至 细胞内。 构建携带编码人共济失调 -毛细血管扩张症突变蛋白 15 的多核苷酸的 重组病毒载体的方法可见于已有文献（Sambrook, e t a l. )。 另外重组编码人共济 失调 -毛细血管扩张症突变蛋白 15 的多核苷酸可包装到脂质体中转移至细胞 内。  Polynucleotides encoding human ataxia-capillary telangiectasia mutant protein 15 can also be used for a variety of therapeutic purposes. Gene therapy techniques can be used to treat abnormalities in cell proliferation, development, or metabolism caused by human ataxia-capillary telangiectasia mutant protein 15 expression or abnormal / inactive expression. Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutant human ataxia-capillary telangiectasia mutant protein 15 to inhibit endogenous human ataxia-capillary telangiectasia mutant protein 1 5 active. For example, a variant human ataxia-telangiectasia mutant protein 15 may be a shortened human ataxia-telangiectasia mutant protein 15 that lacks a signaling domain, although it may interact with downstream substrates. Binding, but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of ataxia-telangiectasia mutant protein 15. 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 ataxia-capillary telangiectasia mutant protein 15 into cells . A method for constructing a recombinant viral vector carrying a polynucleotide encoding a human ataxia-capillary telangiectasia mutant protein 15 can be found in the existing literature (Sambrook, et al.). In addition, a polynucleotide encoding human ataxia-capillary telangiectasia mutant protein 15 can be packaged into liposomes and transferred into cells.
多核苷酸导入组织或细胞内的方法包括： 将多核苷酸直接注入到体内组织 中； 或在体外通过载体（如病毒、 噬菌体或质粒等）先将多核苷酸导入细胞中， 再将细胞移植到体内等。  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.
抑制人共济失调 -毛细血管扩张症突变蛋白 1 5 mRNA的寡核苷酸（包括反义 RNA和 DM)以及核酶也在本发明的范围之内。 核酶是一种能特异性分解特定 A 的酶样 RM分子， 其作用机制是核酶分子与互补的靶 RNA 特异性杂交后进行核 酸内切作用。 反义的 RNA和 DNA及核酶可用已有的任何 RNA或 DNA合成技术获 得， 如固相磷酸酰胺化学合成法合成寡核苷酸的技术已广泛应用。 反义 RNA 分 子可通过编码该 RM 的 DNA序列在体外或体内转录获得。 这种 DNA序列已整合 到载体的 RM 聚合酶启动子的下游。 为了增加核酸分子的稳定性， 可用多种方 法对其进行修饰， 如增加两侧的序列长度， 核糖核苷之间的连接应用磷酸硫酯 键或肽键而非磷酸二酯键。 Oligonucleotides (including antisense RNA and DM) and ribozymes that inhibit human ataxia-telangiectasia mutant protein 15 mRNA are also within the scope of the present invention. A ribozyme is an enzyme-like RM molecule that can specifically decompose a specific A. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target RNA and performs endonucleation. Antisense RNA, DNA, and ribozymes can be obtained by any existing RNA or DNA synthesis technology, such as the technology for the synthesis of oligonucleotides by solid-phase phosphoramidite chemical synthesis has been widely used. Antisense RNA analysis The daughter can be obtained by in vitro or in vivo transcription of the DNA sequence encoding the RM. This DNA sequence has been integrated downstream of the RM polymerase promoter of the vector. In order to increase the stability of a nucleic acid molecule, it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the ribonucleoside linkages should use phosphate thioester or peptide bonds instead of phosphodiester bonds.
编码人共济失调 -毛细血管扩张症突变蛋白 1 5的多核苷酸可用于与人共济 失调 -毛细血管扩张症突变蛋白 15 的相关疾病的诊断。 编码人共济失调 -毛细 血管扩张症突变蛋白 15 的多核苷酸可用于检测人共济失调 -毛细血管扩张症突 变蛋白 1 5的表达与否或在疾病状态下人共济失调 -毛细血管扩张症突变蛋白 1 5 的异常表达。 如编码人共济失调 -毛细血管扩张症突变蛋白 1 5的 DNA序列可用 于对活检标本进行杂交以判断人共济失调 -毛细血管扩张症突变蛋白 1 5 的表达 状况。 杂交技术包括 Southern 印迹法， Nor thern 印迹法、 原位杂交等。 这些 技术方法都是公开的成熟技术， 相关的试剂盒都可从商业途径得到。 本发明的 多核苷酸的一部分或全部可作为探针固定在微阵列（M i croarray)或 DNA芯片（又 称为 "基因芯片" ）上， 用于分析组织中基因的差异表达分析和基因诊断。 用人 共济失调 -毛细血管扩张症突变蛋白 15 特异的引物进行 RNA -聚合酶链反应 (RT-PCR)体外扩增也可检测人共济失调 -毛细血管扩张症突变蛋白 15 的转录产 物。  A polynucleotide encoding human ataxia-capillary telangiectasia mutant protein 15 can be used for diagnosis of diseases related to human ataxia-capillary telangiectasia mutant protein 15. A polynucleotide encoding human ataxia-capillary telangiectasia mutant protein 15 can be used to detect the expression of human ataxia-capillary telangiectasia mutant protein 15 or human ataxia-capillary telangiectasia in a disease state Aberrant Expression of Mutant Protein 1-5. For example, a DNA sequence encoding human ataxia-capillary telangiectasia mutant protein 15 can be used to hybridize biopsy specimens to determine the expression of human ataxia-capillary telangiectasia mutant protein 15. Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly available and mature, and related kits are commercially available. Some or all of the polynucleotides of the present invention can be used as probes to be fixed on a microarray or a DNA chip (also known as a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues. . Human ataxia-telangiectasia mutant protein 15 specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect the human ataxia-telangiectasia mutant protein 15 transcription product.
检测人共济失调 -毛细血管扩张症突变蛋白 1 5基因的突变也可用于诊断人 共济失调 -毛细血管扩张症突变蛋白 15相关的疾病。 人共济失调 -毛细血管扩 张症突变蛋白 15 突变的形式包括与正常野生型人共济失调 -毛细血管扩张症突 变蛋白 1 5 DNA序列相比的点突变、 易位、 缺失、 重组和其它任何异常等。 可用 已有的技术如 Southern 印迹法、 DNA序列分析、 PCR 和原位杂交检测突变。 另 夕卜， 突变有可能影响蛋白的表达， 因此用 Nor thern 印迹法、 Wes t ern 印迹法可 间接判断基因有无突变。  Detection of human ataxia-capillary telangiectasia mutant protein 15 mutations can also be used to diagnose human ataxia-capillary telangiectasia mutant protein 15-related diseases. Human ataxia-capillary telangiectasia mutant protein 15 mutant forms include point mutations, translocations, deletions, recombinations and any other mutations compared to normal wild-type human ataxia-capillary telangiectasia mutant protein 1 5 DNA sequences Exception, etc. Mutations can be detected using existing techniques such as Southern blotting, DNA sequence analysis, PCR and in situ hybridization. In addition, mutations may affect protein expression. Therefore, the Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
本发明的序列对染色体鉴定也是有价值的。 该序列会特异性地针对某条人 染色体具体位置且并可以与其杂交。 目前， 需要鉴定染色体上的各基因的具体 位点。 现在， 只有很少的基于实际序列数据（重复多态性）的染色体标记物可用 于标记染色体位置。 根据本发明， 为了将这些序列与疾病相关基因相关联， 其 重要的第一步就是将这些 DNA序列定位于染色体上。  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. 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.
简而言之， 根据 cDNA制备 PCR引物（优选 15-35bp) , 可以将序列定位于染色 体上。 然后， 将这些引物用于 PCR筛选含各条人染色体的体细胞杂合细胞。 只 有那些含有相应于引物的人基因的杂合细胞会产生扩增的片段。  In short, a PCR primer (preferably 15-35bp) is prepared from the cDNA, and the sequence can be located on the chromosome. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
体细胞杂合细胞的 PCR定位法， 是将 DNA定位到具体染色体的快捷方法。 使 用本发明的寡核苷酸引物， 通过类似方法， 可利用一组来自特定染色体的片段 或大量基因组克隆而实现亚定位。 可用于染色体定位的其它类似策略包括原位 杂交、 用标记的流式分选的染色体预筛选和杂交预选， 从而构建染色体特异的 cDNA库。 PCR localization of somatic hybrid cells is a quick way to localize DNA to specific chromosomes. Make Using the oligonucleotide primers of the present invention, sublocalization can be achieved by a similar method using a set of fragments from a specific chromosome or a large number of genomic clones. Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct chromosome-specific cDNA libraries.
将 cDNA克隆与中期染色体进行荧光原位杂交（FISH) , 可以在一个步骤中精 确地进行染色体定位。 此技术的综述， 参见 Verma等， Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988)。  Fluorescent in situ hybridization (FISH) of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988).
一旦序列被定位到准确的染色体位置， 此序列在染色体上的物理位置就可 以与基因图数据相关联。 这些数据可见于例如， V.Mckusick, Mendel ian Inheritance in Man (可通过与 Johns Hopkins University Welch Medical Library联机获得）。 然后可通过连锁分析， 确定基因与业已定位到染色体区域 上的疾病之间的关系。  Once the sequence is located at the exact chromosomal location, the physical location of the sequence on the chromosome can be correlated with the genetic map data. These data can be found in, for example, V. Mckusick, Mendel ian 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.
接着， 需要测定患病和来患病个体间的 cDNA或基因组序列差异。 如果在一 些或所有的患病个体中观察到某突变， 而该突变在任何正常个体中未观察到， 则该突变可能是疾病的病因。 比较患病和来患病个体， 通常涉及首先寻找染色 体中结构的变化， 如从染色体水平可见的或用基于 cDNA序列的 PCR可检测的缺 失或易位。 根据目前的物理作图和基因定位技术的分辨能力， 被精确定位至与 疾病有关的染色体区域的 cDNA, 可以是 50至 500个潜在致病基因间之一种（假定 1兆碱基作图分辨能力和每 20kb对应于一个基因）。  Next, the cDNA or genomic sequence differences between the affected and the affected 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 diseased individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the 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).
可以将本发明的多肽、 多核苷酸及其模拟物、 激动剂、 拮抗剂和抑制剂与 合适的药物载体组合后使用。 这些载体可以是水、 葡萄糖、 乙醇、 盐类、 缓冲 液、 甘油以及它们的组合。 组合物包含安全有效量的多肽或拮抗剂以及不影响 药物效果的载体和赋形剂。 这些组合物可以作为药物用于疾病治疗。  The polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier. These carriers 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.
本发明还提供含有一种或多种容器的药盒或试剂盒， 容器中装有一种或多 种本发明的药用组合物成分。 与这些容器一起， 可以有由制造、 使用或销售药 品或生物制品的政府管理机构所给出的指示性提示， 该提示反映出生产、 使用 或销售的政府管理机构许可其在人体上施用。 此外， 本发明的多肽可以与其它 的治疗化合物结合使用。  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. Along with 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. In addition, the polypeptides of the invention can be used in combination with other therapeutic compounds.
药物组合物可以以方便的方式给药， 如通过局部、 静脉内、 腹膜内、 肌内、 皮下、 鼻内或皮内的给药途径。 人共济失调 -毛细血管扩张症突变蛋白 15以有 效地治疗和 /或预防具体的适应症的量来给药。 施用于患者的人共济失调 -毛细 血管扩张症突变蛋白 15的量和剂量范围将取决于许多因素， 如给药方式、 待治 疗者的健康条件和诊断医生的判断。  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 ataxia-telangiectasia mutant protein 15 is administered in an amount effective to treat and / or prevent a specific indication. The amount and range of human ataxia-capillary vasodilator mutein 15 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.

Claims

权 利 要 求 书 Claim
1、 一种分离的多肽-人共济失调 -毛细血管扩张症突变蛋白 15, 其特征在于 它包含有： SEQ ID NO: 2 所示的氨基酸序列的多肽、 或其多肽的活性片段、 类 似物或衍生物。 1. An isolated polypeptide-human ataxia-capillary telangiectasia mutant protein 15, characterized in that it comprises: a polypeptide having the amino acid sequence shown in SEQ ID NO: 2 or an active fragment or analog of the polypeptide Or derivatives.
2、 如权利要求 1 所述的多肽， 其特征在于所述多肽、 类似物或衍生物的氨基 酸序列具有与 SEQ ID NO: 2所示的氨基酸序列至少 95%的相同性。  2. The polypeptide according to claim 1, characterized in that the amino acid sequence of the polypeptide, analog or derivative has at least 95% identity with the amino acid sequence shown in SEQ ID NO: 2.
3、 如权利要求 2 所述的多肽， 其特征在于它包含具有 SEQ ID NO: 2 所示的氨 基酸序列的多肽。  3. The polypeptide according to claim 2, further comprising a polypeptide having an amino acid sequence represented by SEQ ID NO: 2.
4、 一种分离的多核苷酸， 其特征在于所述多核苷酸包含选自下组中的一种：4. An isolated polynucleotide, characterized in that said polynucleotide comprises one selected from the group consisting of:
(a) 编码具有 SEQ ID NO: 2 所示氨基酸序列的多肽或其片段、 类似物、 衍生 物的多核苷酸； (a) a polynucleotide encoding a polypeptide having an amino acid sequence shown in SEQ ID NO: 2 or a fragment, analog, or derivative thereof;
(b) 与多核苷酸 （a ) 互补的多核苷酸； 或  (b) a polynucleotide complementary to polynucleotide (a); or
(c) 与 （a ) 或 （b ) 有至少 70%相同性的多核苷酸。  (c) A polynucleotide that is at least 70% identical to (a) or (b).
5、 如权利要求 4 所述的多核苷酸， 其特征在于所述多核苷酸包含编码具有 SEQ ID NO: 2所示氨基酸序列的多核苷酸。 5. The polynucleotide according to claim 4, wherein the polynucleotide comprises a polynucleotide encoding an amino acid sequence represented by SEQ ID NO: 2.
6、如权利要求 4所述的多核苷酸，其特征在于所述多核苷酸的序列包含有 SEQ ID NO: 1 中 1007-1258位的序列或 SEQ ID NO: 1中 1-1462位的序列。  6. The polynucleotide according to claim 4, characterized in that the sequence of the polynucleotide comprises the sequence of positions 1007-1258 in SEQ ID NO: 1 or the sequence of positions 1-1462 in SEQ ID NO: 1. .
7、 一种含有外源多核苷酸的重组载体， 其特征在于它是由权利要求 4-6 中的 任一权利要求所述多核苷酸与质粒、 病毒或运载体表达载体构建而成的重组载 体。  7. A recombination vector containing an exogenous polynucleotide, characterized in that it is a recombination constructed by the polynucleotide according to any one of claims 4-6 and a plasmid, virus or a carrier expression vector Carrier.
8、 一种含有外源多核苷酸的遗传工程化宿主细胞， 其特征在于它是选自于下 列一种宿主细胞：  8. A genetically engineered host cell containing an exogenous polynucleotide, characterized in that it is selected from one of the following host cells:
(a) 用权利要求 7所述的重组载体转化或转导的宿主细胞； 或  (a) a host cell transformed or transduced with the recombinant vector of claim 7; or
(b) 用权利要求 4-6 中的任一权利要求所述多核苷酸转化或转导的宿主细 胞。  (b) a host cell transformed or transduced with a polynucleotide according to any one of claims 4-6.
9、 一种具有人共济失调 -毛细血管扩张症突变蛋白 15活性的多肽的制备方法， 其特征在于所述方法包括：  9. A method for preparing a polypeptide having human ataxia-capillary telangiectasia mutant protein 15 activity, characterized in that the method comprises:
(a) 在表达人共济失调 -毛细血管扩张症突变蛋白 15 条件下， 培养权利要 求 8所述的工程化宿主细胞；  (a) culturing the engineered host cell according to claim 8 under the condition of expressing human ataxia-capillary telangiectasia mutant protein 15;
(b) 从培养物中分离出具有人共济失调 -毛细血管扩张症突变蛋白 15 活性 的多肽。  (b) A polypeptide having human ataxia-capillary telangiectasia mutant protein 15 activity is isolated from the culture.
10、 一种能与多肽结合的抗体，其特征在于所述抗体是能与人共济失调 -毛细 血管扩张症突变蛋白 15特异性结合的抗体。 10. An antibody capable of binding to a polypeptide, characterized in that the antibody is capable of ataxia with humans-capillary Vasodilator mutein 15 specifically binds to antibodies.
11、 一类模拟或调节多肽活性或表达的化合物， 其特征在于它们是模拟、 促进、 拮抗或抑制人共济失调 -毛细血管扩张症突变蛋白 15的活性的化合物。  11. A class of compounds that mimic or regulate the activity or expression of a polypeptide, characterized in that they are compounds that mimic, promote, antagonize or inhibit the activity of human ataxia-capillary telangiectasia mutant protein 15.
12、 如权利要求 11 所述的化合物， 其特征在于它是 SEQ ID NO: 1 所示的多核 苷酸序列或其片段的反义序列。  12. The compound according to claim 11, characterized in that it is an antisense sequence of a polynucleotide sequence or a fragment thereof as shown in SEQ ID NO: 1.
13、 一种权利要求 11 所述化合物的应用， 其特征在于所述化合物用于调节人 共济失调 -毛细血管扩张症突变蛋白 15在体内、 体外活性的方法。  13. The use of the compound according to claim 11, characterized in that the compound is used for a method for regulating the activity of human ataxia-capillary telangiectasia mutant protein 15 in vivo and in vitro.
14、 一种检测与权利要求 1-3 中的任一权利要求所述多肽相关的疾病或疾病易 感性的方法， 其特征在于其包括检测所述多肽的表达量， 或者检测所述多肽的 活性， 或者检测多核苷酸中引起所述多肽表达量或活性异常的核苷酸变异。 14. A method for detecting a disease or susceptibility to a disease associated with a polypeptide according to any one of claims 1-3, characterized in that it comprises detecting the expression level of the polypeptide, or detecting the activity of the polypeptide Or detecting a nucleotide variation in a polynucleotide that causes abnormal expression or activity of the polypeptide.
15、 如权利要求 1- 3 中的任一权利要求所述多肽的应用， 其特征在于它应用于 筛选人共济失调 -毛细血管扩张症突变蛋白 15 的模拟物、 激动剂， 拮抗剂或 抑制剂； 或者用于肽指紋图谱鉴定。 15. Use of the polypeptide according to any one of claims 1 to 3, characterized in that it is used for screening a mimetic, agonist, antagonist or inhibitor of human ataxia-capillary telangiectasia mutant protein 15. Agents; or for identification of peptide fingerprints.
16、 如权利要求 4-6 中的任一权利要求所述的核酸分子的应用， 其特征在于它 作为引物用于核酸扩增反应， 或者作为探针用于杂交反应， 或者用于制造基因 芯片或微阵列。  16. The use of a nucleic acid molecule according to any one of claims 4-6, characterized in that it is used as a primer for a nucleic acid amplification reaction, or as a probe for a hybridization reaction, or for manufacturing a gene chip Or microarray.
17、 如权利要求 1-6 及 11 中的任一权利要求所述的多肽、 多核苷酸或化合物 的应用， 其特征在于用所述多肽、 多核苷酸或其模拟物、 激动剂、 拮抗剂或抑 制剂以安全有效剂量与药学上可接受的载体组成作为诊断或治疗与人共济失调 -毛细血管扩张症突变蛋白 15异常相关的疾病的药物组合物。  17. Use of a polypeptide, polynucleotide or compound according to any one of claims 1-6 and 11, characterized in that the polypeptide, polynucleotide or mimetic, agonist, antagonist is used Or the inhibitor is composed of a safe and effective dose with a pharmaceutically acceptable carrier as a pharmaceutical composition for diagnosing or treating a disease associated with human ataxia-telangiectasia mutant protein 15 abnormality.
18、 权利要求 1-6 及 1 1 中的任一权利要求所述的多肽、 多核苷酸或化合物的 应用， 其特征在于用所述多肽、 多核苷酸或化合物制备用于治疗如恶性肿瘤， 血液病， H I V感染和免疫性疾病和各类炎症的药物。  18. Use of a polypeptide, polynucleotide or compound according to any one of claims 1 to 6 and 1 1, characterized in that the polypeptide, polynucleotide or compound is used for preparing a treatment such as a malignant tumor, Hematological diseases, HIV infection and immune diseases and drugs of various inflammations.
PCT/CN2001/000230 2000-03-02 2001-02-26 A novel polypeptide - human ataxia-telangiectasia mutant protein 15 and a polynucleotide encoding the same WO2001074995A2 (en)

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CN 00111825 CN1311219A (en) 2000-03-02 2000-03-02 New polypeptide-human ataxia-telangiectasis mutation protein 15 and polynucleotide for coding said polypeptide
CN00111825.0 2000-03-02

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CLIN. GENET. vol. 55, no. 5, 1999, pages 289 - 304 *
HUM. MUTAT. vol. 10, no. 2, 1997, pages 100 - 107 *
INT. J. BIOCHEM. CELL. BIOL. vol. 31, no. 7, 1999, pages 735 - 740 *
J. CLIN. IMMUNOL. vol. 16, no. 5, 1996, pages 254 - 260 *

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