WO2001040294A1 - Nouveau polypeptide, thrombospondine humaine 30, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, thrombospondine humaine 30, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001040294A1
WO2001040294A1 PCT/CN2000/000494 CN0000494W WO0140294A1 WO 2001040294 A1 WO2001040294 A1 WO 2001040294A1 CN 0000494 W CN0000494 W CN 0000494W WO 0140294 A1 WO0140294 A1 WO 0140294A1
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polypeptide
human
polynucleotide
protein
sequence
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PCT/CN2000/000494
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English (en)
French (fr)
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Yumin Mao
Yi Xie
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Bioroad Gene Development Ltd. Shanghai
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Priority to AU16875/01A priority Critical patent/AU1687501A/en
Publication of WO2001040294A1 publication Critical patent/WO2001040294A1/zh

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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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention belongs to the field of biotechnology. Specifically, the present invention describes a new polypeptide, human thromboembolism 30, and a polynucleotide sequence encoding the polypeptide. The invention also relates to a preparation method and application of the polynucleotide and the polypeptide. Background technique
  • THBS Throrabospondin
  • THBS includes at least the following four functional domains: the amino-terminal heparin-binding domain; type I repeats containing the CSVTCG sequence: type III containing the RGDA sequence III calcium ion-binding repeats; the cell or platelet-binding domain at the carboxyl terminus.
  • the experimental results show that the C00H-terminal cell-binding domain of THBS contains at least two sequences (GRGDSP and VTCG), which can regulate the binding of THBS to cells or platelets. (J Biol Chera 1992 Aug 15; 267 (23): 16230-6)
  • THBS contains domains that bind to heparin, glucosinolate, fibrinogen, fibronectin, fibrinolysin, and type V collagen.
  • THBS1 contains many different functional domains, including three repeating subunits (types I, ⁇ , III).
  • the specific research results show that: The type I repeat structure is encoded by symmetrical exons; the heparin binding domain is encoded by a single exon.
  • the type I repeat structure is encoded by symmetrical exons; the heparin binding domain is encoded by a single exon.
  • THBS The main function of THBS is to cause platelet aggregation.
  • the glycoprotein Ilb-IIIa complex located on the membrane of THBS functions as a fibrinogen receptor. When it binds to fibrinogen, the first reversible agglutination occurs. Under stronger stimulation, the coagulation Thromboprotein is released from the alpha particles of platelets, and through its interaction with fibrinogen, it will lead to the second step of irreversible platelet aggregation.
  • THBS1 is a subfamily of thromboembolic proteins. In vitro experiments show that THBS1 can regulate cell adhesion, proliferation, migration, and differentiation. In vivo, the function of THBS1 has been studied through recombination experiments of homologous genes. The results show that THBS1 mutations can lead to platelet deficiency, but thrombin-induced Platelet aggregation did not decrease. THBS1-deficient mice have no obvious mutations in the brain, heart, kidney, spleen, stomach, intestine, arteries, liver and other tissues, but only abnormalities are found in the THBS1-deficient lung, which can lead to the occurrence of pneumonia and the number of macrophages. Reduction.
  • Thromboprotein has an important role in the healing process of skin lesions. In addition, it has effects on the fibrosing diseases of the avascular tissue (lens) of the eye. Treatment is also extremely important. (Histol Histopathol 1999 Oct; 14 (4): 1309-20)
  • THBS1 is a sticky glycoprotein that can promote the adhesion of human breast cancer cells to human vascular endothelial cells, while heparin-binding domains and type I and II repeating structural domains can regulate the aforementioned effect.
  • the polypeptide of the present invention was inferred and identified as a novel human thromboembolic protein 30 (HTHBS30), the homologous protein of which is the type 1 thromboembolic protein of Mus musculus, and the protein number is AB016768.
  • HTHBS30 human thromboembolic protein 30
  • the human thromboprotein 30 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 it has been necessary in the art to identify more people involved in these processes Thromboprotein 30 protein, especially the amino acid sequence of this protein is identified.
  • the isolation of the new human thromboembolic protein 30 gene also provides a basis for research to determine its role in health and disease states. This protein may form the basis for developing diagnostic and / or therapeutic drugs for the disease, so it is important to isolate its coding for DM. Disclosure of invention
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide a method for producing human thromboembolic protein 30.
  • Another object of the present invention is to provide an antibody against the polypeptide of the present invention-human thromboembolic protein 30.
  • Another object of the present invention is to provide mimic compounds, antagonists, agonists, and inhibitors of human thromboprotein 30 in response to the polypeptide of the present invention. It is another object of the present invention to provide a method for diagnosing and treating diseases associated with abnormalities of human thromboprotein 30.
  • the present invention relates to an isolated polypeptide, which is of human origin, and includes: a polypeptide having the amino acid sequence of SEQ ID D. 2, or a conservative variant, biologically active fragment, or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 322-1140 in SEQ ID NO: 1; and (b) a sequence having 1-2429 in SEQ ID NO: 1 Sequence of bits.
  • the invention further relates to a vector, in particular an expression vector, containing the polynucleotide of the invention; a host cell genetically engineered with the vector, including a transformed, transduced or transfected host cell; and a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • a vector in particular an expression vector, containing the polynucleotide of the invention
  • a host cell genetically engineered with the vector including a transformed, transduced or transfected host cell
  • a method comprising culturing said Host cell and method of preparing the polypeptide of the present invention by recovering the expression product.
  • the invention also relates to an antibody capable of specifically binding to a polypeptide of the invention.
  • the present invention also relates to a method for screening compounds that mimic, activate, antagonize or inhibit the activity of human thromboprotein 30 protein, which comprises utilizing the polypeptide of the present invention.
  • the invention also relates to compounds obtained by this method.
  • the invention also relates to a method for in vitro detection of a disease or susceptibility to disease associated with abnormal expression of human thromboembol protein 30, which comprises detecting a mutation in the polypeptide or a sequence encoding a polynucleotide thereof in a biological sample, or detecting a biological sample The amount or biological activity of a polypeptide of the invention.
  • 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 use of the polypeptide and / or polynucleotide of the present invention for the preparation of a medicament for treating cancer, developmental disease or immune disease or other diseases caused by abnormal expression of human thromboembol 30.
  • Nucleic acid sequence refers to an oligonucleotide, a nucleotide or a polynucleotide and a fragment or part thereof, and may also refer to a genomic or synthetic DNA or RNA, they can be single-stranded or double-stranded, representing the sense or antisense strand.
  • amino acid sequence refers to an oligopeptide, peptide, polypeptide or protein sequence and fragments or portions thereof.
  • amino acid sequence in the present invention relates to the amino acid sequence of a naturally occurring protein molecule, such "polypeptide” or “protein” does not mean to limit the amino acid sequence to a complete natural amino acid related to the protein molecule .
  • a protein or polynucleotide “variant” refers to an amino acid sequence having one or more amino 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 substituted amino acid has a structural or chemical property similar to the original amino acid, such as the replacement of isoleucine with leucine. Variants can also have non-conservative changes, such as replacing glycine with tryptophan.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • Insertion means that a change in the amino acid sequence or nucleotide sequence results in an increase in one or more amino acids or nucleotides compared to a molecule that exists in nature.
  • Replacement refers to the replacement of one or more amino acids or nucleotides with different amino acids or nucleotides.
  • Bioactivity refers to a protein that has the structure, regulation, or biochemical function of a natural molecule.
  • immunologically active refers to the ability of natural, recombinant or synthetic proteins and fragments thereof to induce a specific immune response and to bind specific antibodies in a suitable animal or cell.
  • An "agonist” refers to a molecule that, when combined with human thromboembol 30, causes a change in the protein to regulate the activity of the protein.
  • An agonist may include a protein, a nucleic acid, a carbohydrate, or any other molecule that binds human thromboprotein 30.
  • Antagonist refers to a molecule that can block or regulate the biological or immunological activity of human thromboprotein 30 when combined with human thromboprotein 30.
  • Antagonists and inhibitors may include proteins, nucleic acids, carbohydrates, or any other molecule that can bind human thromboprotein 30.
  • Regular refers to a change in the function of human thromboprotein 30, including an increase or decrease in protein activity, a change in binding properties, and any other biological, functional, or immune changes in human thromboprotein 30.
  • substantially pure means substantially free of other proteins, lipids, carbohydrates or other substances with which it is naturally associated.
  • Those skilled in the art can purify human thromboprotein 30 using standard protein purification techniques. Essentially pure human thromboprotein 30 produces a single main band on a non-reducing polyacrylamide gel. The purity of the human coagulation thrombin 30 polypeptide can be analyzed by amino acid sequence.
  • Complementary refers to polynucleotides that naturally bind through base-pairing under conditions of acceptable salt concentration and temperature.
  • sequence CGA
  • GACT complementary sequence
  • the complementarity between two single-stranded molecules may be partial or complete.
  • the degree of complementarity between nucleic acid strands The efficiency and strength of inter-strand hybridization have a significant effect.
  • “Homology” refers to the degree of complementarity and can be partially homologous or completely homologous.
  • Partial homology refers to a partially complementary sequence that at least partially inhibits hybridization of a fully complementary sequence to a target nucleic acid. Inhibition of such hybridization can be detected by performing hybridization (Southern blotting or Nor thern blotting, etc.) under conditions of reduced stringency. Substantially homologous sequences or hybridization probes can compete and inhibit binding of completely homologous sequences to the target sequence under conditions of reduced stringency. This does not mean that the conditions of reduced stringency allow non-specific binding, because the conditions of reduced stringency require that the two sequences be combined with each other as a specific or selective interaction.
  • Percent identity refers to the percentage of sequences that are the same or similar in the comparison of two or more amino acid or nucleic acid sequences. The percent identity can be determined electronically, such as through the MEGALIGN program (Lasergene sof tware package, DNASTAR, Inc., Madis on Wi s.). The MEGALIGN program can compare two or more sequences according to different methods, such as the Cluster method (Higg ins, DG and PM Sharp (1988) Gene 73: 237-244). The distance between the two groups of sequences is arranged into clusters. Then the clusters are allocated 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: Number of matching residues
  • the number of residues in sequence A-the number of spacer residues in sequence A-the number of spacer residues in sequence B can also be determined by the Cluster method or by methods known in the art such as Jotun He in. Sex percentage (He in J., (1990) Methods in emzumo l ogy 183: 625-645) 0
  • Similarity refers to the degree of identical or conservative substitutions of amino acid residues at corresponding positions in the alignment of amino acid sequences.
  • Amino acids used for conservative substitutions for example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; having an uncharged head group is Similar hydrophilic amino acids may include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; serine and threonine; phenylalanine and tyrosine.
  • Antisense refers to a nucleotide sequence that is complementary to a particular DM or RNA sequence. “Antisense strand” means
  • Sense strand A complementary nucleic acid strand.
  • Derivative refers to a chemical modification of HFP or a nucleic acid encoding it. Such a chemical modification may be the replacement of a hydrogen atom with an alkyl group, an acyl group or an amino group. Nucleic acid derivatives can encode polypeptides that retain the main biological characteristics of natural molecules.
  • Antibody refers to a complete antibody molecule and its fragments, such as Fa,? ( ⁇ ') 2 and? ⁇ Its specificity An epitope that binds human thromboprotein 30.
  • 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 matter from its original environment (for example, its natural environment if it is naturally occurring).
  • a naturally occurring polynucleotide or polypeptide is not isolated when it is present in a living animal, but the same polynucleotide or polypeptide is separated from some or all of the substances that coexist in the natural system.
  • Such a polynucleotide may be part of a vector, or such a polynucleotide or polypeptide may be part of a composition. Since the carrier or composition is not part of its natural environment, they are still isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and peptides in the natural state of living cells are not isolated and purified, but the same polynucleotides or peptides are separated and purified if they are separated from other substances existing in the natural state. .
  • isolated human thromboprotein 30 means that human thromboprotein 30 is substantially free of other proteins, lipids, sugars, or other substances with which it is naturally associated. Those skilled in the art can purify human thromboprotein 30 using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel. The purity of human thromboprotein 30 polypeptide can be analyzed by amino acid sequence.
  • the present invention provides a new polypeptide, human thrombus protein 30, which is basically composed of the amino acid sequence shown in SEQ ID NO: 2.
  • the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide.
  • the polypeptide of the present invention may be a naturally purified product or a chemically synthesized product, or produced using recombinant technology from a prokaryotic or eukaryotic host (eg, bacteria, yeast, higher plants, insects, and mammalian cells) according to a recombinant production scheme
  • a prokaryotic or eukaryotic host eg, bacteria, yeast, higher plants, insects, and mammalian cells
  • the host used, the polypeptide of the present invention may be glycosylated, or may be non-glycosylated.
  • the polypeptides of the invention may also include or exclude the starting methionine residue.
  • the invention also includes fragments, derivatives, and analogs of human thromboprotein 30.
  • fragment refers to a polypeptide that substantially maintains the same biological function or activity of the human thromboembolic protein 30 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 the genetic code; or (II) a type in which a group on one or more amino acid residues is replaced by another group and contains a substituent: or ( ⁇ ⁇ )
  • Another compound such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol
  • polypeptide sequences in which additional amino acid sequences are fused into mature polypeptides such as leader sequences or secreted sequences or sequences used to purify this polypeptide or proteinogen sequences
  • the present invention provides an isolated nucleic acid (polynucleotide), which basically consists of a polynucleotide encoding a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the polynucleotide sequence of the present invention includes a nucleotide sequence of SEQ ID NO: 1.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It contains a polynucleotide sequence of 2429 bases in length and its open reading frame (322-1140) encodes 272 amino acids. According to the amino acid sequence homology comparison, it was found that this polypeptide has 47% homology with the type 1 coagulation protein of Mus musculus. It can be inferred that the human thromboembolic protein 30 has a similar structure and function of the type 1 coagulation protein of Mus musculus .
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDM, genomic DNA or synthetic DNA ...
  • DNA can be single-stranded or double-stranded.
  • DNA can be coding or non-coding.
  • the coding region sequence encoding a mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or a degenerate variant.
  • degenerate variant means in the present invention that the code has A protein or polypeptide of SEQ ID NO: 2 but a nucleic acid sequence different 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 that includes the polypeptide and a polynucleotide that includes 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.
  • This polynucleotide variant can be a naturally occurring allelic variant or a non-naturally occurring variant.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide, which may be one or more nucleotides Substitutions, deletions, or insertions without substantially altering the function of the polypeptide it encodes.
  • the invention also relates to a polynucleotide that hybridizes to the sequence described above (having at least 50%, preferably 70% identity between the two sequences).
  • the present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions.
  • stringent conditions means: (1) hybridization at lower ionic strength and higher temperature And elution, such as 0.2xSSC, 0.1% SDS, 60 ° C; or (2) adding a denaturant during hybridization, such as 50% ( ⁇ / ⁇ ) formamide, 0.1% calf serum / 0 l% F i co ll, 42.
  • polypeptide encoded by the crossed polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2.
  • nucleic acid fragments that hybridize to the sequences described above.
  • a "nucleic acid fragment” contains at least 10 nucleotides in length, preferably at least 20-30 nucleotides, more preferably at least SO-SO nucleotides, most preferably at least 100 cores. Glycylic acid or more. Nucleic acid fragments can also be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding human thromboembolic protein 30.
  • 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 thrombus protein 30 of the present invention can be obtained by various methods.
  • polynucleotides are isolated using hybridization techniques well known in the art. These techniques include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect homologous polynucleotide sequences, and 2) antibody screening of expression libraries to detect cloned polynucleosides with common structural characteristics Acid fragments. ⁇
  • the DNA fragment sequence of the present invention can also be obtained by the following methods: 1) isolating the double-stranded DNA sequence from the genomic DNA; 2) chemically synthesizing the DNA sequence to obtain the double-stranded DNA of the polypeptide.
  • genomic DNA isolation is the least commonly used. Direct chemical synthesis of DNA sequences is often the method of choice. The more commonly used method is the isolation of cDNA sequences.
  • the standard method for isolating the cDNA of interest is to isolate mRNA from donor cells that overexpress the gene and perform reverse transcription to form a plasmid or phage cDNA library.
  • Various methods have been used to extract mRNA, and kits are also commercially available (Qiagene).
  • 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 combined with polymerase reaction technology, even very small expression products can be cloned.
  • genes of the present invention can be selected from these cDNA libraries by conventional methods. These methods include (but are not limited to): (l) DNA-DNA or DNA-RNA hybridization; (2) the presence or absence of a marker gene function; (3) the determination of the level of the human coagulation protein 30 transcript; (4) Detection of gene-expressed protein products by immunological techniques or determination of biological activity. The above methods can be used singly or in combination.
  • the probe used for hybridization is homologous to any part of the polynucleotide of the present invention, and its length is at least 10 nucleotides, preferably at least 30 nucleotides, more preferably At least 50 nucleotides, preferably at least 100 nucleotides.
  • the length of the probe is usually within 2000 nucleotides, preferably within 1000 nucleotides.
  • the probe used here is 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 the human thrombus 30 gene expression can be detected by immunological techniques such as Western blotting, radioimmunoprecipitation, and enzyme-linked immunosorbent assay (ELISA).
  • a method for amplifying MA / RNA by PCR (Saiki, et al. Science 1985; 230: 1350-1354) is preferably used to obtain the gene of the present invention.
  • the RACE method RACE-rapid amplification of cDNA ends
  • the primers used for PCR may be appropriately based on the polynucleotide sequence information of the present invention disclosed herein. Select and synthesize using conventional methods.
  • the amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.
  • polynucleotide sequence of the gene of the present invention or various DNA fragments and the like obtained as described above can be determined by a conventional method such as dideoxy chain termination method (Sanger et al. PNAS, 1977, 74: 5463-5467). Such polynucleotide sequences can also be determined using commercial sequencing kits and the like. In order to obtain the full-length cDNA sequence, the sequencing must be repeated. Sometimes it is necessary to determine the cDNA sequence of multiple clones in order to splice into a full-length cDNA sequence.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, and a host cell that is genetically engineered using the vector of the present invention or directly using a human thromboprotein 30 coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology.
  • the polynucleotide sequence encoding human thromboembolic protein 30 may 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.
  • the expression vector also includes a ribosome binding site and a transcription terminator for translation initiation. Insertion of enhancer sequences into the vector will enhance its transcription in higher eukaryotic cells. Enhancers are cis-acting factors for DNA expression, usually about 10 to 300 base pairs, which act on the promoter To enhance gene transcription. Illustrative examples include SV40 enhancers of 100 to 270 base pairs on the late side of the origin of replication, polyoma enhancers on the late side of the origin of replication, and adenovirus enhancers.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture.
  • GFP fluorescent protein
  • tetracycline or ampicillin resistance for E. coli.
  • a polynucleotide encoding human thrombus protein 30 or a recombinant vector containing the polynucleotide can be transformed or transduced into a host cell to constitute a genetically engineered host cell containing the polynucleotide or the recombinant vector.
  • host cell refers to a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • Escherichia coli, Streptomyces bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells insect cells
  • fly S2 or Sf 9 animal cells
  • animal cells such as CH0, COS or Bowes melanoma cells.
  • Transformation of a host cell with a DNA sequence described in the present invention or a recombinant vector containing the DNA sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of 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 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 thromboembolic protein 30 (Scence, 1984; 224: 1431). Generally, the following steps are taken:
  • the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.
  • a suitable method such as temperature conversion or chemical induction
  • the recombinant polypeptide may be coated in a cell, expressed on a cell membrane, or secreted outside the cell.
  • recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to: conventional renaturation treatment, protein precipitant treatment (salting out method), centrifugation, osmotic disruption, ultrasound Processing, 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, ultrasound Processing, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC
  • Fig. 1 is a comparison diagram of amino acid sequence homology between human thrombus protein 30 and type 1 clotting protein of Mus musculus.
  • the upper sequence is human thromboembol 30, and the lower sequence is type 1 clotting protein from Mus musculus.
  • Identical amino acids are represented by single-character amino acids between the two sequences, and similar amino acids are represented by "+”.
  • Figure 2 shows the polyacrylamide gel electrophoresis (SDS-PAGE) of human thromboembol 30 isolated.
  • 30kDa is the molecular weight of the protein.
  • the arrow indicates the isolated protein band.
  • RNA from human fetal brain was extracted by guanidine isothiocyanate / phenol / chloroform method. Using QuikmRNA Isolation Kit
  • the determined cDNA sequence was compared with the existing public DNA sequence database (Genebank), and the cDN A sequence of one of the clones, 1009 A 07, was found to be new DNA.
  • a series of primers were synthesized to determine the inserted cDNA fragments of the clone in both directions.
  • the results showed that the 1009A07 clone contained a full-length cDNA of 2429bp (as shown in Seq ID N0: 1), and an 819bp open reading frame (0RF) from 322bp to 1140bp, encoding a new protein (such as Seq ID NO : Shown in 2).
  • This clone PBS-1009A07 and the encoded protein was named human thromboembolic protein 30.
  • Example 2 Homologous search of cDNA clones
  • the sequence of the human thromboembolic protein 30 of the present invention and the protein sequence encoded by the same are subjected to the Blast program (Basiclocal Alignment search tool) [Altschul, SF et al. J. Mol. Biol. 1990; 215: 403-10], Homology search in databases such as Genbank and Swissport.
  • the gene with the highest homology to the human thromboembolic protein 30 of the present invention is a known type 1 coagulation protein of Mus musculus, and its accession number to Genbank is AB016768.
  • the protein homology results are shown in Figure 1. The two are highly homologous with 47% identity; 64% similarity.
  • Example 3 Cloning of a Gene Encoding Human Thrombin 30 by RT-PCR
  • CDNA was synthesized using fetal brain total RNA as a template and oligo-dT as a primer.
  • PCR amplification was performed with the following primers:
  • Primerl 5,-AGGGAACAAAAGCTGGAGCTCCAC -3, (SEQ ID NO: 3)
  • Primer2 5'- AGGCCCATGTGTCTATAATTAGAT -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 reaction conditions containing 50mmol / L KC1 in a reaction volume of 50 ⁇ 1, 10mmol / L Tris - CI, (P H8.5), 1.5mmol / L MgCl 2, 200 ⁇ mol / L dNTP, lOpmoi primer 1U Taq polymerase (product of Clontech), reacted on a PE9600 DNA thermal cycler (Perkin-Elmer) for 25 cycles under the following conditions: 94 ° C 30sec; 55 ° C 30sec; 72. C 2min.
  • This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49: 1) are added. ), Mix and centrifuge. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate. The resulting RNA pellet was washed with 70% ethanol, dried and dissolved in water.
  • 32P-labeled probe (about 2 x 10 6 cpm / ml) was hybridized with a nitrocellulose membrane to which RNA was transferred overnight in a solution containing 50% formamide-25raM KH 2 P0 4 (pH7.4)-5 x SSC- 5 x Denhardt, s solution and 200 ⁇ ⁇ / ⁇ 1 salmon sperm DNA. After hybridization, the filters were placed in 1 X SSC-0.1% SDS at 55 ° C. C for 30 min. Then, Phosphor Imager was used for analysis and quantification.
  • Example 5 In vitro expression, isolation and purification of recombinant human thromboembolic protein 30
  • Primer3 5'- CATGCTAGCATGCACTTGCGACTGATTTCTTGGC -3 '(Seq ID No: 5)
  • Primer4 5'- CATGGATCCCTAGTGTACAGTGCTGACTGATACC -3, (Seq ID No: 6)
  • the 5' ends of these two primers contain Nhel and BamHI restriction sites, respectively.
  • the coding sequences of the 5 'and 3' ends of the gene of interest are followed, respectively.
  • the Nhel and BamHI restriction sites correspond to the selectivity within the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865.3). Digestion site.
  • PCR was performed using the PBS-1009A07 plasmid containing the full-length target gene as a template.
  • the PCR reaction conditions were as follows: a total volume of 50 ⁇ l contained 10 pg of BS-1009A07 plasmid, primers Primer-3 and Primer-4 were lpmol, Advantage polymerase Mix (Clontech) 1 ⁇ 1, respectively. Cycle parameters: 94 ° C 20s, 60. C 30s, 68 ° C 2 min, 25 cycles in total. Nhel and BamHI were used to double digest the amplified product and plasmid pET-28 (+), respectively, and large fragments were recovered and ligated with T4 ligase. The ligation product was transformed into E.
  • coli DH5a by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 g / ml), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-1009A07) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) P lySs (product of Novagen) using the calcium chloride method. In LB liquid medium containing kanamycin (final concentration 30 g / ml), the host bacteria BL21 (pET-1009A07) was cultured at 37 ° C to the logarithmic growth phase, and IPTG was added to a final concentration of 1 mmol / L.
  • a peptide synthesizer (product of PE company) was used to synthesize the following human thrombolytic protein 30-specific peptides: NH 2 -Met-His-Leu-Arg-Leu-I le-Ser-Trp-Leu-Phe-I le-I le-Leu-Asn-Phe-C00H (SEQ ID NO: 7).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • hemocyanin and bovine serum albumin For methods, see: Avrameas, et al. Immunochemistry, 1969; 6: 43. 4mg of the above hemocyanin polypeptide complex With complete Freund's adjuvant immunization, 15 days later the hemocyanin polypeptide complex plus incomplete Freund's adjuvant was used to boost the immunity once.
  • EL using a 15 g / ml bovine serum albumin peptide complex-coated titer plate for EL I SA to determine antibody titers in rabbit serum. Total I gG was isolated from antibody-positive rabbit serum using protein A-Sepharose.
  • the peptide was bound to a cyanogen bromide-activated Se P ha r 0 S e4B column, and the antibody was separated from total I gG by affinity chromatography. Polypeptide antibodies. The immunoprecipitation method demonstrated that the purified antibody could specifically bind to human thromboprotein 30. Industrial applicability
  • 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.
  • the polypeptide (human thromboprotein 30) of the present invention is a glycoprotein, which is a component of al pha particles of platelets.
  • the main function of human thromboprotein 30 of the present invention is to cause platelet aggregation, except that in platelets,
  • the synthesis and secretion of human thrombus protein 30 is also related to endothelial cells, fibroblasts, and smooth muscle cells.
  • Human thrombus protein 30 can also regulate cell adhesion, proliferation, migration, and differentiation. Studies have found that thrombin has an important role in the healing process of skin injuries. In addition, thrombin plays an extremely important role in the treatment of ocular non-vascular tissue (lens) fibrosis.
  • the human thromboembolic protein 30 of the present invention can be used for the diagnosis and treatment of many diseases, including: blood system diseases, malignant tumors, endocrine system diseases, neurological diseases, immune diseases, human acquired immune deficiency syndrome (A IDS), etc. Wait,
  • the hematological diseases that the human thrombus protein 30 of the present invention can use for treatment are mainly hemorrhagic diseases, including: abnormalities or qualitative platelets such as idiopathic thrombocytopenic purpura, thrombocytosis, and thrombocytosis; coagulation dysfunction Such as lack of various coagulation factors (such as hemophilia), multi-factor hemorrhagic diseases such as disseminated intravascular coagulation, increased circulation of anticoagulant substances; abnormalities of blood vessel walls such as hereditary hemorrhagic capillary dilatation, allergic Purpura vascular purpura; thrombosis and so on.
  • hemorrhagic diseases including: abnormalities or qualitative platelets such as idiopathic thrombocytopenic purpura, thrombocytosis, and thrombocytosis; coagulation dysfunction Such as lack of various coagulation factors (such as hemophilia), multi-factor hemorrhagic diseases such as disse
  • the human thrombus protein 30 of the present invention also has a certain effect on hematological diseases caused by other reasons.
  • the following examples are specific diseases that can be treated with the human thromboprotein 30 of the present invention:
  • Hemorrhagic diseases hereditary capillary dilatation, allergic purpura vascular purpura, simple purpura, idiopathic thrombocytopenic purpura. Hemophilia, vascular hemophilia;
  • liver disease DK
  • thrombotic diseases transfusion and transfusion reactions, etc.
  • Developmental disorders that can be treated with the human thromboembolic protein 30 of the present invention include: spina bifida, craniocerebral fissure, anencephaly deformity, cerebral bulge, foramen deformity, Down syndrome, congenital hydrocephalus, aqueduct malformation, Cartilage hypoplasia dwarfism, spinal epiphyseal dysplasia, pseudochondral dysplasia, Langer- G ied i on syndrome, funnel chest, gonad hypoplasia, congenital adrenal hyperplasia, upper urethral fissure, cryptic, with short stature syndrome (such as Conrad i syndrome and Danbo l tC l os s syndrome), Congenital glaucoma or cataracts, congenital lens abnormalities, congenital blepharoplasia, retinal dysplasia, congenital optic nerve atrophy, congenital sensorineural hearing loss, cracked hands and feet, teratosis, Wi
  • Various tumors that can be treated with the human thromboembolic protein 30 of the present invention include: including epithelial tissues (such as basal epithelium, squamous epithelium, mucus cells, etc.), (such as fibrous tissue, adipose tissue, cartilage tissue, smooth muscle tissue, blood vessels And lymphatic endothelial tissue, etc.), hematopoietic tissue (such as B cells, T cells, tissue cells, etc.), central nervous tissue, peripheral nervous tissue, endocrine tissue, gonadal tissue, special tissue (such as dental tissue, etc.)
  • epithelial tissues such as basal epithelium, squamous epithelium, mucus cells, etc.
  • fibrous tissue such as fibrous tissue, adipose tissue, cartilage tissue, smooth muscle tissue, blood vessels And lymphatic endothelial tissue, etc.
  • hematopoietic tissue such as B cells, T cells, tissue cells, etc.
  • central nervous tissue such as
  • the human thromboembolic protein 30 of the present invention is also an immunomodulator, and has an immune promoting or immunosuppressing effect.
  • the polypeptide of the present invention can be used for the treatment of diseases including non-response of immune response, or abnormal immune response, or ineffective host defense.
  • the polypeptides and antibodies of the present invention also have effects on damage, defects or disorders of immune tissues, especially for diseases of the hematopoietic system (such as malignant anemia), skin diseases (such as psoriasis), and autoimmune diseases (such as rheumatoid arthritis). ), Radiation diseases and the production and regulation of immune lymphocytes are extremely closely related.
  • the invention also provides methods of screening compounds to identify agents that increase (agonist) or suppress (antagonist) human thromboprotein 30.
  • Agonists enhance biological functions such as human thrombus protein 30 to stimulate cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or a membrane preparation expressing human thromboprotein 30 can be cultured with labeled human thromboprotein 30 in the presence of a drug. The ability of the drug to increase or block this interaction is then determined.
  • Antagonists of human thromboprotein 30 include selected antibodies, compounds, receptor deletions, and the like. Antagonists of human thromboprotein 30 can bind to human thromboprotein 30 and eliminate its function, or inhibit the production of the polypeptide, or bind to the active site of the polypeptide so that the polypeptide cannot perform biological functions.
  • human thromboprotein 30 When screening compounds as antagonists, human thromboprotein 30 can be added to a bioanalytical assay to determine whether the compound is an antagonist by measuring the effect of the compound on the interaction between human thromboprotein 30 and its receptor. 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 thromboprotein 30 can be obtained by screening a random peptide library composed of various possible combinations of amino acids bound to a solid phase. When screening, 30 molecules of human thromboprotein are generally labeled.
  • the present invention provides the use of polypeptides, and fragments, derivatives, analogs or cells thereof as antigens.
  • Methods of producing antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the human thrombus 30 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 obtained by directly injecting immunized animals (such as rabbits, mice, rats, etc.) with human thromboprotein 30.
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies to human thromboembolic protein 30 include, but are not limited to, hybridoma technology (Kohler and Milstei n. 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 (Morris on e t a l, PNAS, 1 985, 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 thromboprotein 30.
  • Antibodies to human thromboprotein 30 can be used in immunohistochemical techniques to detect human thromboprotein 30 in biopsy specimens.
  • Monoclonal antibodies that bind to human thromboprotein 30 can also be labeled with radioisotopes and injected into the body to track their location and distribution. This radiolabeled antibody can be used as a non-invasive diagnostic method to locate tumor cells and determine whether there is metastasis.
  • Antibodies can also be used to design immunotoxins that target a particular part of the body.
  • human thromboprotein 30 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 cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds.
  • This hybrid antibody can be used to kill human thromboembolic protein 30 positive cells.
  • the antibodies of the present invention can be used to treat or prevent diseases related to human thromboembol. Administration of an appropriate dose of the antibody can stimulate or block the production or activity of human thromboprotein 30.
  • the present invention also relates to a diagnostic test method for quantitative and localized detection of human thromboembolic protein 30 levels.
  • tests are well known in the art and include FI SH assays and radioimmunoassays.
  • the level of human thromboprotein 30 detected in the test can be used to explain the importance of human thromboprotein 30 in various diseases and to diagnose diseases in which human thromboprotein 30 plays a role.
  • polypeptide of the present invention can also be used for peptide mapping analysis.
  • the polypeptide can be specifically cleaved by physical, chemical or enzymatic analysis, and subjected to one-dimensional or two-dimensional or three-dimensional gel electrophoresis analysis, and more preferably mass spectrometry analysis.
  • Polynucleotides encoding human thromboprotein 30 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 the non-expression or abnormal / inactive expression of human thromboprotein 30.
  • Recombinant gene therapy vectors (such as viral vectors) can be designed to express mutated human thromboprotein 30 to inhibit endogenous human thromboprotein 30 activity.
  • a variant human thromboprotein 30 may be a shortened human thromboprotein 30 lacking a signaling domain. The substrate binds but lacks signaling activity. Therefore, the recombinant gene therapy vector can be used to treat diseases caused by abnormal expression or activity of human thromboembolic protein 30.
  • Virus-derived expression vectors such as retrovirus, adenovirus, adenovirus-associated virus, herpes simplex virus, parvovirus and the like can be used to transfer a polynucleotide encoding human thromboprotein 30 into cells.
  • a method for constructing a recombinant viral vector carrying a polynucleotide encoding human thrombus protein 30 can be found in the existing literature (Sambrook, etai.).
  • a recombinant polynucleotide encoding human thromboprotein 30 can be packaged into liposomes and transferred into cells.
  • Methods for introducing a polynucleotide into a tissue or cell include: directly injecting the polynucleotide into a tissue in vivo; or introducing the polynucleotide into a cell in vitro through a vector (such as a virus, phage, or plasmid), and then transplanting the cell Into the body and so on.
  • a vector such as a virus, phage, or plasmid
  • Oligonucleotides including antisense RNA and DNA
  • ribozymes that inhibit human thromboprotein 30 mRNA are also within the scope of the present invention.
  • the core enzyme is an enzyme-like RNA molecule that specifically breaks down specific RNAs, and its mechanism of action It is a ribozyme molecule that specifically hybridizes with a complementary target RNA for endonucleation.
  • Antisense RNA, DNA, and ribozymes can be obtained using any existing RNA or DNA synthesis technology, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides.
  • Antisense RNA molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA.
  • This DM sequence has been integrated downstream of the RNA polymerase promoter of the vector.
  • it can be modified in a variety of ways, such as increasing the sequence length on both sides, and the linkage between ribonucleosides using phosphate thioester or peptide bonds instead of phosphodiester bonds.
  • a polynucleotide encoding human thromboprotein 30 can be used for the diagnosis of diseases related to human thromboprotein 30.
  • the polynucleotide encoding human thromboprotein 30 can be used to detect the expression of human thromboprotein 30 or the abnormal expression of human thromboprotein 30 in a disease state.
  • a DNA sequence encoding human thrombus protein 30 can be used to hybridize biopsy specimens to determine the expression of human thrombus protein 30.
  • Hybridization techniques include Southern blotting, Nor thern blotting, and in situ hybridization. These techniques and methods are publicly known and mature, and related kits are commercially available.
  • a part or all of the polynucleotides of the present invention can be used as probes to be fixed on a micro array or a DNA chip (also called a "gene chip") for analyzing differential expression analysis and gene diagnosis of genes in tissues.
  • Human thromboprotein 30 specific primers can also be used to detect the transcription products of human thromboprotein 30 by RNA-polymerase chain reaction (RT-PCR) in vitro amplification.
  • RT-PCR RNA-polymerase chain reaction
  • Detection of mutations in the human thrombus protein 30 gene can also be used to diagnose human thrombus protein 30-related diseases.
  • the form of the human thrombus 30 mutation includes point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type human thromboprotein 30 DNA sequence. 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, Nor thern blotting and Western blotting can be used to indirectly determine whether a gene is mutated.
  • sequences of the invention are also valuable for chromosome identification. This sequence will be specific to someone The chromosome is in a specific location and can be crossed with 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 labeling 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.
  • the PCR primers (preferably 15-35b P ) are prepared based on 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 hybrid cells that contain the human gene corresponding to the primer will produce amplified fragments.
  • PCR localization of somatic hybrid cells is a quick way to localize DM to specific chromosomes.
  • oligonucleotide primers of the present invention in a similar manner, a set of fragments from a specific chromosome or a large number of genomic clones can be used to achieve sublocalization.
  • Other similar strategies that can be used for chromosomal localization include in situ hybridization, chromosome pre-screening with labeled flow sorting, and hybrid pre-selection to construct a chromosome-specific c library.
  • 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 affected and unaffected individuals usually involves first looking for structural changes in chromosomes, such as deletions or translocations that are visible at the chromosomal level or detectable with cDNA sequence-based PCR. According to the resolution capabilities of current physical mapping and gene mapping technology, the 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 that do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • the present invention also provides a kit or kit containing one or more containers containing one or more ingredients of the pharmaceutical composition of the present invention.
  • containers there can be medicines manufactured or used by or sold An indicative prompt given by a government regulatory agency for biological products that reflects the permission of the government regulatory agency that produces, uses, or sells it to be administered to the human body.
  • the polypeptides of the invention can be used in combination with other therapeutic compounds.
  • the pharmaceutical composition can be administered in a convenient manner, such as by a topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal route of administration.
  • Human thromboprotein 30 is administered in an amount effective to treat and / or prevent a specific indication.
  • the amount and range of human thromboprotein 30 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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Description

一种新的多肽一一人凝血栓蛋白 30和编码这种多肽的多核苷酸 技术领域
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凝 血栓蛋白 30， 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
凝血栓蛋白 （ throrabospondin, THBS ) 是一种糖蛋白， 它是血小板的 alpha 颗粒的组分， 在血小板被激活后释放， 在二价阳离子存在时 THBS 结合于血小板 膜， 它可以帮助血小板的凝聚。 除了在血小板中有作用外， THBS 的合成、 分泌 还与内皮细胞、 纤维原细胞、 平滑肌细胞等都有关系。 （ J Cell Biol 1982 Oct; 95 (1) 351-4 )
THBS至少包括以下四种功能域： 氨基末端的肝磷脂结合功能域； 包含 CSVTCG 序列的类型 I重复片段： 包含 RGDA 序列的类型 III钙离子结合重复片段； 羧基末 端的细胞或血小板结合功能域。 实验的结果表明， THBS 的 C00H-末端的细胞结合 功能域包含有至少两段序列 （ GRGDSP和 VTCG ) ， 可以调控 THBS与细胞或血小板 的结合。 ( J Biol Chera 1992 Aug 15; 267 (23)： 16230-6 )
从结构上看， THBS 包含有可与肝磷脂、 硫苷脂、 纤维蛋白原、 纤维结合蛋 白、 纤维蛋白溶解原、 类型 V胶原质等结合的结构域。
根据原位杂交的结果， 编码人 THBS1 的基因被定位于人染色体 15ql5, THBS1 包含有很多不同的功能域， 包括三个重复的亚基 （类型 I， Π, III ) , 具体的研 究结果表明： 类型 I重复结构由对称的外显子编码； 肝磷脂结合功能域由一个单 个的 外 显 子编 码 ， 具体的 结构请 参阅相 关 文献 。 （ Genomics 1990 Apr; 6 (4)： 685-91 )
THBS的主要功能是导致血小板的凝聚。 位于 THBS的膜上的糖蛋白 Ilb-IIIa 复合物是作为纤维蛋白原受体起作用的， 当其与纤维蛋白原结合时， 就发生第一 步可逆的凝集， 在更强的刺激下， 凝血栓蛋白从血小板的 alpha颗粒上释放出来， 通过其与纤维蛋白原的相互作用， 会导致第二步不可逆的血小板凝集。 （Amui Rev Med 1986; 37: 179-86 )
THBS1 是凝血栓蛋白的一个亚家族。 体外的实验结果表明， THBS1 可以调控 细胞的粘合、 增殖、 移动和分化。 在体内， THBS1 的功能通过同源基因的重组实 验得以研究， 结果表明， THBS1 的变异会导致血小板的缺乏， 但是凝血酶诱导的 血小板凝聚反应并未减少。 THBS1缺乏的鼠的脑、 心脏、 肾、 脾、 胃、 肠、 动脉、 肝等组织并未有明显的变异， 只是在缺乏 THBS1 的肺中发现了异常， 会导致肺炎 的发生和巨噬细胞数目的减少。 ( J Clin Invest 1998 Mar 1; 101 (5): 982-92 ) 在皮肤损伤的治愈过程中， 凝血栓蛋白有着重要的作用， 此外， 它对眼部无 血管组织 （ 晶状体） 纤维化的疾病的治疗也有着极其重要的作用。 （ Histol Histopathol 1999 Oct; 14 (4)： 1309-20 )
还有研究表明， THBS1 是一种有粘性的糖蛋白， 它可以促进人乳癌细胞与人 的血管内皮细胞的粘合， 而肝磷脂结合功能域和类型 I, II重复结构功能域可以 调节上述的作用。 ( J Cell Biochem 1996 Sep 15; 62 (4): 431-42 )
根据氨基酸同源比较的结果， 本发明的多肽被推断鉴定为一种新的人凝血栓 蛋白 30 ( HTHBS30 ) ， 其同源蛋白是小家鼠的类型 1 凝血栓蛋白， 蛋白号是 AB016768. 由于如上所述人凝血栓蛋白 30 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凝血栓蛋白 30 蛋白， 特别是鉴定这种蛋白的 氨基酸序列。 新人凝血栓蛋白 30 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病诊断和 /或治疗 药的基础， 因此分离其编码 DM是非常重要的。 发明的公开
本发明的一个目的是提供分离的新的多肽一一人凝血栓蛋白 30 以及其片 段、 类似物和衍生物。
本发明的另一个目的是提供编码该多肽的多核苷酸。
本发明的另一个目的是提供含有编码人凝血栓蛋白 30 的多核苷酸的重组载 体。
本发明的另一个目的是提供含有编码人凝血栓蛋白 30 的多核苷酸的基因工 程化宿主细胞。
本发明的另一个目的是提供生产人凝血栓蛋白 30的方法。
本发明的另一个目的是提供针对本发明的多肽一一人凝血栓蛋白 30 的抗 体。
本发明的另一个目的是提供了针对本发明多肽一一人凝血栓蛋白 30 的模拟 化合物、 拮抗剂、 激动剂、 抑制剂。 本发明的另一个目的是提供诊断治疗与人凝血栓蛋白 30 异常相关的疾病的 方法。
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 ID NO: 2氨基酸序列的多肽。
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：
(a)编码具有 SEQ I D No. 2氨基酸序列的多肽的多核苷酸；
(b)与多核苷酸（a)互补的多核苷酸；
(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。
更佳地， 该多核苷酸的序列是选自下组的一种： （a)具有 SEQ ID NO: 1 中 322- 1140位的序列； 和（b)具有 SEQ ID NO: 1 中 1 -2429位的序列。
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。
本发明还涉及一种能与本发明多肽特异性结合的抗体。
本发明还涉及一种筛选的模拟、 激活、 拮抗或抑制人凝血栓蛋白 30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。
本发明还涉及一种体外检测与人凝血栓蛋白 30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。
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。
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癌症、 发育性疾 病或免疫性疾病或其它由于人凝血栓蛋白 30 表达异常所引起疾病的药物的用 途。
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 DNA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。
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。
"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。
"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。
"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。
"激动剂" 是指当与人凝血栓蛋白 30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 它可结合人凝血栓蛋白 30的分子。
"拮抗剂" 或 "抑制物" 是指当与人凝血栓蛋白 30结合时， 一种可封闭或调 节人凝血栓蛋白 30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凝血栓蛋白 30的分子。
"调节" 是指人凝血栓蛋白 30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凝血栓蛋白 30的任何其它生物学性质、 功能或免疫性质 的改变。
"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凝血栓蛋白 30。 基本 上纯的人凝血栓蛋白 30 在非还原性聚丙烯酰胺凝胶上能产生单一的主带。 人凝 血栓蛋白 30多肽的纯度可用氨基酸序列分析。
"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。
"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 （ Southern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。
"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 MEGALIGN程序 （Las ergene sof tware package, DNASTAR, Inc. , Mad i s on Wi s. ) 。 MEGALIGN程序可根据不同 的方法如 C l us t er法比较两种或多种序列（H igg ins, D. G. 和 P. M. Sharp (1988) Gene 73: 237-244)„ C lus 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 1 00 序列 A的残基数-序列 A中间隔残基数-序列 B中间隔残基数 也可以通过 C lus ter法或用本领域周知的方法如 Jotun He in 测定核酸序列之 间的相同性百分率（He in J. , (1990) Methods in emzumo l ogy 183: 625-645) 0
"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。
"反义" 是指与特定的 DM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。
"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。
"抗体" 是指完整的抗体分子及其片段， 如 Fa、 ？(^') 2及？^ 其能特异性 结合人凝血栓蛋白 30的抗原决定簇。
"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。
"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。 多如本发明所用， "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。
如本文所用， "分离的人凝血栓蛋白 30" 是指人凝血栓蛋白 30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凝血栓蛋白 30。 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。 人凝血栓蛋白 30多肽的纯度能用氨基酸序列分析。
本发明提供了一种新的多肽一一人凝血栓蛋白 30,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。
本发明还包括人凝血栓蛋白 30 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凝血栓蛋白 30 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 （ Π Ι ) 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、 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。
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 N0: 1 的核 苷酸序列。 本发明的多核苷酸是从人胎脑组织的 cDNA 文库中发现的。 它包含的 多核苷酸序列全长为 2429 个碱基， 其开放读框 （ 322-1140 ) 编码了 272 个氨基 酸。 根据氨基酸序列同源比较发现， 此多肽与小家鼠的类型 1 凝血蛋白有 47%的 同源性， 可推断出该人凝血栓蛋白 30 具有小家鼠的类型 1 凝血蛋白相似的结构 和功能。
本发明的多核苷酸可以是 DNA形式或是 RNA形式。 DNA形式包括 cDM、 基因 组 DNA 或人工合成的 DNA ... DNA 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 ID 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。
编码 SEQ ID NO: 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。
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。
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。
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% (ν/ν)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。
本发明还涉及与以上所描述的序列杂交的核酸片段。 如本发明所用， "核酸 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 SO- SO 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 /或分离编码人凝血栓蛋白 30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。 本发明的编码人凝血栓蛋白 30 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。 ·
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 DNA序列以获得所述多肽的双链 DNA。
上述提到的方法中， 分离基因组 DNA最不常用。 DNA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 C lontech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。
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- DNA 或 DNA- RNA 杂交； （2)标志基因功能的出现或丧失； （3)测定人 凝血栓蛋白 30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。
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。
在第（4)种方法中. 检测人凝血栓蛋白 30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（ELISA)等。 应 用 PCR 技 术 扩 增 MA/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 DNA/RNA片段。
如上所述得到的本发明的基因， 或者各种 DNA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。
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 人凝血栓蛋白 30 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。
本发明中， 编码人凝血栓蛋白 30 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 MSXND 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。
本领域的技术人员熟知的方法能用于构建含编码人凝血栓蛋白 30 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 DNA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)。 所述的議序歹1 j 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 DNA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。
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。
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。
本发明中， 编码人凝血栓蛋白 30 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。
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。
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 组的人凝血栓蛋白 30 (Sc i ence , 1984 ; 224 : 1431)。 一般来说有以下歩骤：
(1) .用本发明的编码人 人凝血栓蛋白 30 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；
(2) .在合适的培养基中培养宿主细胞；
(3) .从培养基或细胞中分离、 纯化蛋白质。
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。
在步骤 （ 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
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。
图 1是本发明人凝血栓蛋白 30和小家鼠的类型 1凝血蛋白的氨基酸序列同源 性比较图。 上方序列是人凝血栓蛋白 30， 下方序列是小家鼠的类型 1凝血蛋白。 相同氨基酸在两个序列间用单字符氨基酸表示， 相似氨基酸用 "+" 表示。
图 2为分离的人凝血栓蛋白 30的聚丙烯酰胺凝胶电泳图（SDS- PAGE)。 30kDa 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
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。
实施例 1: 人凝血栓蛋白 30的克隆
用异硫氰酸胍 /酚 /氯仿一歩法提取人胎脑总 RNA。 用 QuikmRNA 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 cDNA片段定向插入到 pBSK (+)载体（Clontech公司产品）的多克隆位点上， 转化 DH5oc , 细菌形成 cDNA文 库。 用 Dye terminate cycle react ion sequencing kit (Perkin-Elmer公司产品 ) 和 ABI 377自动测序仪（Perkin- Elmer公司）测定所有克隆的 5'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 1009 A 07的 cDN A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， 1009A07克隆所含的全长 cDNA为 2429bp (如 Seq ID N0: 1所示 ） , 从第 322bp至 1140bp有一个 819bp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-1009A07, 编码的蛋白质命名为人凝血栓蛋白 30。 实施例 2: cDNA 克隆的同源检索 将本发明的人凝血栓蛋白 30的序列及其编码的蛋白序列， 用 Blast程序 (Bas iclocal Alignment search tool) [Altschul, SF et al. J.Mol. Biol.1990; 215: 403-10] , 在 Genbank、 Swissport等数据库进行同源检索。 与本发明的人凝血栓蛋白 30同源性最高的基因是一种已知的小家鼠的类型 1凝血 蛋白， 其编码的蛋白在 Genbank的准入号为 AB016768。 蛋白质同源结果示于图 1， 两者高度同源， 其相同性为 47%; 相似性为 64%。 实施例 3: 用 RT-PCR方法克隆编码人凝血栓蛋白 30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
Qiagene的试剂盒纯化后，用下列引物进行 PCR扩增：
Primerl: 5，- AGGGAACAAAAGCTGGAGCTCCAC -3， (SEQ ID NO: 3)
Primer2: 5'- AGGCCCATGTGTCTATAATTAGAT -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；
Primer2为 SEQ ID NO: 1的中的 3'端反向序列。
扩增反应的条件： 在 50 μ 1的反应体积中含有 50mmol/L KC1, 10mmol/L Tris - CI, (PH8.5) , 1.5mmol/L MgCl 2, 200 μ mol/L dNTP, lOpmoi引物， 1U的 Taq 醒聚 合酶（Clontech公司产品） , 在 ΡΕ9600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 - actin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 Unvitrogen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- 2429bp完全相同。 实施例 4: Northern 印迹法分析人凝血栓蛋白 30基因的表达：
用一步法提取总 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 20 gRNA， 在含 20mM3- (N - 吗啉代） 丙磺酸 （ pH7.0 ) -5mM乙酸钠 _lmM 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 cx- 32P dATP通过随机引物法制备 32P- 标记的 DNA探针。 所用的 I)NA探针为图 1所示的 PCR扩增的人凝血栓蛋白 30编码区序 列（322bp至 1140bp)。 将 32P-标记的探针 (约 2 χ 106cpm/ml ) 与转移了 RNA的硝酸 纤维素膜在一溶液中于 杂交过夜， 该溶液包含 50%甲酰胺 -25raM KH2P04 (pH7.4) - 5 x SSC- 5 x Denhardt，s溶液和 200 μ§/ιη1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 5: 重组人凝血栓蛋白 30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：
Primer3: 5'- CATGCTAGCATGCACTTGCGACTGATTTCTTGGC -3' ( Seq ID No: 5 ) Primer4: 5'- CATGGATCCCTAGTGTACAGTGCTGACTGATACC -3， ( Seq ID No: 6 ) 此两段引物的 5'端分别含有 Nhel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 ， Nhel和 BamHI酶切位点相应于表达载体质粒 pET- 28b (+) (Novagen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-1009A07质粒为模板， 进行 PCR反应。 PCR反应条件为： 总体积 50 μ 1中含 BS-1009A07质粒 10pg、 引物 Primer- 3和 Primer- 4分别为 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 Nhel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 DH5a,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- 1009A07) 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 30 g/ml )的 LB液体培养基中， 宿主菌 BL21( pET- 1009A07 )在 37°C 培养至对数生长期， 加入 IPTG至终浓度 lmmol/L， 继续培养 5小时。 离心收集菌体， 经超声波破菌，离心收集工清， 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 蛋白人凝血栓蛋白 30。 经 SDS-PAGE电泳， 在 30kDa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 6 抗人凝血栓蛋白 30抗体的产生
用多肽合成仪 （PE公司产品 ） 合成下述人凝血栓蛋白 30特异性的多肽： NH2-Met-His-Leu-Arg-Leu-I le-Ser-Trp-Leu-Phe-I le-I le-Leu-Asn-Phe-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免， 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。 釆用经 15 g/ml牛血清白蛋白多肽复合物包被的滴定板做 EL I SA 测定兔血清中抗体的滴度。 用蛋白 A-Sepharose从抗体阳性的家兔血清中分离总 I gG . 将多肽结合于溴化氰活化的 SePha r0 S e4B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凝血栓蛋白 30结合。 工业实用性
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。
本发明的多肽 （人凝血栓蛋白 30 ) 是一种糖蛋白， 它是血小板的 a l pha 颗 粒的组分， 本发明的人凝血栓蛋白 30 的主要功能是导致血小板的凝聚， 除了在 血小板中有作用外， 人凝血栓蛋白 30 的合成、 分泌还与内皮细胞、 纤维原细胞、 平滑肌细胞等都有关系， 人凝血栓蛋白 30 还可以调控细胞的粘合、 增殖、 移动 和分化等过程。 研究发现， 在皮肤损伤的治愈过程中， 凝血栓蛋白有着重要的作 用。 此外， 凝血栓蛋白对眼部无血管组织 （晶状体） 纤维化的疾病的治疗也有着 极其重要的作用。
本发明的人凝血栓蛋白 30 可用于很多疾病的诊断和治疗， 包括： 血液系统 疾病， 恶性肿瘤， 内分泌系统疾病， 神经系统疾病， 免疫性疾病， 人获得性免疫 缺乏综合症 （A IDS ) 等等、
本发明的人凝血栓蛋白 30 可用于治疗的血液系统疾病主要是出血性疾病， 包括： 血小板数量或质的异常如特发性血小板减少性紫癫， 血小板增多症， 血小 板无力症； 凝血功能障碍如各种凝血因子缺乏 （如血友病） ， 多因素所致出血性 疾病如播散性血管内凝血、 循环中抗凝血物质增多； 血管壁异常如遗传性出血性 毛细管扩张症， 过敏性紫癫血管性紫癫； 血栓形成等等。
当然， 本发明的人凝血栓蛋白 30 对于其他原因引起的血液系统疾病也有一 定的作用， 以下举例为具体可用本发明的人凝血栓蛋白 30治疗的疾病：
出血性疾病： 遗传性毛细管扩张症， 过敏性紫癫血管性紫癫， 单纯性紫癫， 特发性血小板减少性紫癫. 血友病， 血管性血友病；
相关血液病有： 肝脏疾病， DK , 血栓性疾病， 输血和输血反应等等。
利用本发明的人凝血栓蛋白 30 可以治疗的发育紊乱症包括： 脊柱裂、 颅脑 裂、 无脑畸形、 脑膨出、 孔脑畸形、 Down 综合症、 先天性脑积水、 导水管畸形、 软骨发育不全性侏儒病、 脊柱骨骺发育不良症、 假软骨发育不全症、 Langer - G ied i on 综合症、 漏斗胸、 生殖腺发育不全、 先天性肾上腺增生、 尿道上裂、 隐 、 伴有身材矮小的畸形综合症（如 Conrad i综合症与 Danbo l t-C l os s综合症）、 先天性青光眼或白内障、 先天性晶状体位置异常、 先天性小睑裂、 视网膜发育异 常、先天性视神经萎缩、先天性感觉神经性听觉损失、裂手裂脚症、畸胎、 Wi l l i ams 综合症、 A l ag i l l e综合症、 贝魏二氏综合症等等。
利用本发明的人凝血栓蛋白 30可以治疗的各种肿瘤包括： 包括上皮组织（如 基底上皮、 鳞形上皮、 粘液细胞等等） 、 （如纤维组织、 脂肪组织、 软骨组织、 平滑肌组织、 血管和淋巴管内皮组织等等） 、 造血组织 （如 B细胞、 T细胞、 组 织细胞等等） 、 中枢神经组织、 周围神经组织、 内分泌组织、 性腺组织、 特殊组 织 （如牙组织等等） 来源的肿瘤， 例如， 胃癌、 肝癌、 大肠癌、 乳腺癌、 肺癌、 前列腺癌、 宫颈癌、 胰腺癌、 食道癌等等。
本发明的人凝血栓蛋白 30 还是一种免疫调节剂， 具有免疫促进或免疫抑制 作用。 本发明的多肽可用于一些疾病的治疗， 这些疾病包括免疫反应的无反应性， 或异常免疫反应， 或宿主防卫无效。 本发明的多肽和其抗体对免疫组织的损伤、 缺陷或失调类疾病也有作用， 特别是对于造血系统疾病 （如恶性贫血） ， 皮肤病 (如牛皮癣） ， 自身免疫病 （如类风湿性关节炎） ， 放射性疾病以及免疫淋巴细 胞的生成和调节有极为密切的关系。
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）人凝血栓蛋 白 30的药剂的方法。 激动剂提高人凝血栓蛋白 30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凝血栓蛋白 30 的膜制剂与标记的人凝血栓蛋白 30一起培养。 然后测定药物提高或阻遏此相互作用的能力。
人凝血栓蛋白 30 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。 人凝血栓蛋白 30的拮抗剂可以与人凝血栓蛋白 30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。
在筛选作为拮抗剂的化合物时， 可以将人凝血栓蛋白 30 加入生物分析测定 中， 通过测定化合物对人凝血栓蛋白 30 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。 能与人凝血栓蛋白 30 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 凝血栓蛋白 30分子进行标记。
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 对人凝血栓蛋白 30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 Fa b表达文库产生的片段。
多克隆抗体的生产可用人凝血栓蛋白 30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。 制备人凝血栓蛋白 30的单克隆抗体的技术包括但不限于杂交瘤技术（Koh l er and Mi l s t e i n. Na t ur e , 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（Mor r i s on e t a l ， PNAS, 1 985， 81 : 6851)。 而已有的生产单链抗体的技术（U. S. Pa t No. 4946778)也可用于生产抗人凝血栓蛋白 30的单链抗体。
抗人凝血栓蛋白 30 的抗体可用于免疫组织化学技术中， 检测活检标本中的 人凝血栓蛋白 30。
与人凝血栓蛋白 30 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。
抗体还可用于设计针对体内某一特殊部位的免疫毒素。 如人凝血栓蛋白 30 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凝血栓蛋白 30 阳性的细胞。
本发明中的抗体可用于治疗或预防与人凝血栓蛋白 30 相关的疾病。 给予适 当剂量的抗体可以刺激或阻断人凝血栓蛋白 30的产生或活性。
本发明还涉及定量和定位检测人凝血栓蛋白 30 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 凝血栓蛋白 30水平， 可以用作解释人凝血栓蛋白 30在各种疾病中的重要性和用 于诊断人凝血栓蛋白 30起作用的疾病。
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。
编码人凝血栓蛋白 30 的多核苷酸也可用于多种治疗目的。 基因治疗技术可 用于治疗由于人凝血栓蛋白 30 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凝血 栓蛋白 30 , 以抑制内源性的人凝血栓蛋白 30 活性。 例如， 一种变异的人凝血栓 蛋白 30 可以是缩短的、 缺失了信号传导功能域的人凝血栓蛋白 30， 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凝血 栓蛋白 30 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凝血栓蛋 白 30的多核苷酸转移至细胞内。 构建携带编码人凝血栓蛋白 30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 i . )。 另外重组编码人凝血栓 蛋白 30的多核苷酸可包装到脂质体中转移至细胞内。
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。
抑制人凝血栓蛋白 30 mRNA 的寡核苷酸（包括反义 RNA和 DNA)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 RNA的酶样 RNA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 RNA的 DNA序列 在体外或体内转录获得。这种 DM序列已整合到载体的 RNA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。
编码人凝血栓蛋白 30的多核苷酸可用于与人凝血栓蛋白 30的相关疾病的诊 断。 编码人凝血栓蛋白 30的多核苷酸可用于检测人凝血栓蛋白 30的表达与否或 在疾病状态下人凝血栓蛋白 30的异常表达。 如编码人凝血栓蛋白 30的 DNA序列 可用于对活检标本进行杂交以判断人凝血栓蛋白 30 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（Mi 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。 用人凝血栓蛋白 30 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凝血栓蛋白 30 的转录产 物。
检测人凝血栓蛋白 30 基因的突变也可用于诊断人凝血栓蛋白 30 相关的疾 病。 人凝血栓蛋白 30突变的形式包括与正常野生型人凝血栓蛋白 30 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。 另外， 突变有可能影响蛋白 的表达， 因此用 Nor thern 印迹法、 Wes tern印迹法可间接判断基因有无突变。
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。
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。
体细胞杂合细胞的 PCR定位法， 是将 DM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 c醒库。
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)。
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。
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。 比较患病和未患病个体， 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）。
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。
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。
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。 人凝血栓蛋白 30 以有效地治疗和 /或预防具体的 适应症的量来给药。 施用于患者的人凝血栓蛋白 30 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。

Claims

1、 一种分离的多肽 -人凝血栓蛋白 30 , 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。
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— I D NO: 2所示的氨基酸序列至少 95%的相同性。
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。
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：
(a) 编码具有 SEQ I D 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；
(b) 与多核苷酸 （a ) 互补的多核苷酸； 或
(c) 与 （ a ) 或 （ b ) 有至少 70%相同性的多核苷酸。
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 I D N0: 2所示氨基酸序列的多核苷酸。
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 322- 1 140位的序列或 SEQ I D NO: 1 中 1-2429位的序列。
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。
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：
(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
(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。
9、 一种具有人凝血栓蛋白 30 活性的多肽的制备方法， 其特征在于所述方法 包括：
(a) 在表达人凝血栓蛋白 30条件下， 培养权利要求 8所述的工程化宿主细胞；
(b) 从培养物中分离出具有人凝血栓蛋白 30活性的多肽。
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凝血栓蛋白 30特 异性结合的抗体,.
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、 拮抗或抑制人凝血栓蛋白 30的活性的化合物。
12、 如权利要求 1 1 所述的化合物， 其特征在于它是 SEQ I D NO: 1 所示的多核 苷酸序列或其片段的反义序列。
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 凝血栓蛋白 30在体内、 体外活性的方法。
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。
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 筛选人凝血栓蛋白 30 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。
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。
17、 如权利要求 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凝血栓蛋白 30异常相关的疾病的药物组合物。
18、 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、 多核苷酸或化合物制备用于治疗如恶性肿瘤， 血 液病， H IV感染和免疫性疾病和各类炎症的药物。
PCT/CN2000/000494 1999-11-29 2000-11-27 Nouveau polypeptide, thrombospondine humaine 30, et polynucleotide codant pour ce polypeptide WO2001040294A1 (fr)

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AU16875/01A AU1687501A (en) 1999-11-29 2000-11-27 A novel polypeptide - human thrombospondin-30 and a polynucleoti de encoding thesame

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CN 99124148 CN1297930A (zh) 1999-11-29 1999-11-29 一种新的多肽——人凝血栓蛋白30和编码这种多肽的多核苷酸
CN99124148.7 1999-11-29

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001077169A3 (en) * 2000-04-05 2002-07-04 Kirin Brewery Methods and materials relating to stem cell growth factor-like polypeptides and polynucleotides
WO2007100357A3 (en) * 2005-10-07 2009-05-07 Nuvelo Inc Stem cell factor-like protein scfa1 and uses thereof

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0514721A1 (en) * 1991-05-22 1992-11-25 W.R. Grace & Co.-Conn. Peptides having thrombospondin-like activity and their therapeutic use
WO1994013794A1 (en) * 1992-12-04 1994-06-23 Brigham And Women's Hospital, Inc. Human thrombospondin-4

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0514721A1 (en) * 1991-05-22 1992-11-25 W.R. Grace & Co.-Conn. Peptides having thrombospondin-like activity and their therapeutic use
WO1994013794A1 (en) * 1992-12-04 1994-06-23 Brigham And Women's Hospital, Inc. Human thrombospondin-4

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001077169A3 (en) * 2000-04-05 2002-07-04 Kirin Brewery Methods and materials relating to stem cell growth factor-like polypeptides and polynucleotides
WO2007100357A3 (en) * 2005-10-07 2009-05-07 Nuvelo Inc Stem cell factor-like protein scfa1 and uses thereof

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CN1297930A (zh) 2001-06-06
AU1687501A (en) 2001-06-12

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