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WO2001019865A1 - Nouvelle muskeline et ses procedes d'application et de production - Google Patents

Nouvelle muskeline et ses procedes d'application et de production Download PDF

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Publication number
WO2001019865A1
WO2001019865A1 PCT/CN2000/000269 CN0000269W WO0119865A1 WO 2001019865 A1 WO2001019865 A1 WO 2001019865A1 CN 0000269 W CN0000269 W CN 0000269W WO 0119865 A1 WO0119865 A1 WO 0119865A1
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Prior art keywords
polypeptide
polynucleotide
human
thromboprotein
sequence
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PCT/CN2000/000269
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
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Shanghai Biorigin Gene Development Co. Ltd.
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Priority to CN00812875A priority Critical patent/CN1387535A/zh
Publication of WO2001019865A1 publication Critical patent/WO2001019865A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a novel polynucleotide, and a polypeptide encoded by the same; it also relates to a method for producing these polynucleotides and polypeptides, and uses thereof. Specifically, the present invention relates to a nucleotide sequence encoding a novel human thromboprotein-1 mediation factor and the polypeptide. Background technique
  • Thrombospondin-1 mediator (Muskelin) is a newly discovered, widely expressed intracellular thromboprotein-1 regulatory protein that promotes thrombospondin-1 to attach to the cytoskeleton and is a cell Adhesive mediator (EMBO J 1998, Sep 1; 17 (17): 4976-74)
  • Thromboprotein-1 is a glycoprotein that stimulates thrombin released by platelet a-particles. It is also a component of the extracellular matrix required for growth and repair. Its trimer The molecular weight is 420kDa. There are many factors that regulate the expression of TSP-1, and its proteins are degraded by both extracellular and intracellular pathways (Int J Biochem Cell Biol 1997 Jun; 29 (6): 861-5).
  • Thromboprotein-1 mediator mainly exists in the cytoplasm or attaches to the cell membrane, and its function is to stimulate the cell to attach to the C and T terminals of TSP-1.
  • Studies on Drosophila Kelch 0RF1 protein and C2C12 cells have shown that its Function is to stimulate cell attachment to TSP -1 C, end. Deletion of TPS-1 mediators results in reduced cell adhesion, proliferation, and cytoskeletal tissue activity of cells containing TSP-1 attachment agent.
  • thromboprotein-1 Human thromboprotein-1 mediators regulate cell migration, cell proliferation, neurite growth, and angiogenesis through its adhesion to molecules within cells. Studies on the underlying molecular mechanisms of these activities have been conducted It has been shown that thromboprotein-1 is medically useful for hemostasis and angiogenesis.
  • the present invention provides a new mature polypeptide, that is, a human thrombus-1 mediator, and also provides polypeptide fragments, analogs, and derivatives having the same biological activity as the polypeptide.
  • the present invention also provides isolated nucleic acid molecules encoding human thromboprotein-1 mediator, and fragments, analogs, and derivatives of the polynucleotide encoding the same biologically active polypeptide.
  • the invention provides a vector that can comprise a polynucleotide of the invention.
  • the invention provides a host cell comprising a polynucleotide or a vector of the invention.
  • the invention also provides methods of using the polypeptide of the invention to produce antibodies, agonists or antagonists of the polypeptide and the antibodies, agonists and antagonists produced thereby.
  • the invention also provides the use of a polypeptide or polynucleotide of the invention and an antibody, agonist or antagonist in the diagnosis or treatment of a disease. For example, it is used in the diagnosis or treatment of diseases related to tumors, spinal dysplasia, and osteosclerosis.
  • the present invention also provides a pharmaceutical composition comprising the polypeptide, polynucleotide, antibody, agonist or antagonist of the present invention.
  • the present invention provides a novel human thromboprotein-1 mediation factor, and fragments, analogs and derivatives thereof having biological activity.
  • Another aspect of the present invention provides isolated nucleic acid molecules encoding these polypeptides, including mRNA, DNA, cDNA and genomic DNA, and biologically active fragments, analogs and derivatives thereof.
  • the invention provides an isolated polynucleotide comprising a nucleotide sequence of the following group:
  • a preferred polynucleotide of the present invention comprises a polypeptide encoding an amino acid sequence shown in SEQ ID No. 2.
  • a more preferred polynucleotide of the present invention comprises the sequence from positions 24 to 2231 in SEQ ID No. 1.
  • the polynucleotide of the present invention comprises the sequence of positions 1 to 3011 in SEQ ID No. 1.
  • isolated means that the material is extracted from its original environment.
  • a polynucleotide that is present in a living animal is not isolated, but the same polynucleotide that is isolated from some or all of the coexisting materials in the natural system is isolated.
  • Such a polynucleotide may be part of a vector or a composition, as long as the vector or composition is not part of its natural environment, the polynucleotide is still isolated.
  • the polynucleotide of the present invention is found from a cDNA library of human fetal brain tissue. It has the characteristics of a thromboembolic protein-1 mediator in structure. At the same time, it has a high degree of homology with the squirrel thromboprotein-1 mediator. Of the 735 amino acids in the column, 728 proteins are identical, that is, 99% homology.
  • the polynucleotide in the present invention may be in the form of RNA or DNA, and the DNA therein may be cDNA, genomic DNA and synthetic DNA.
  • DNA can be double-stranded or single-stranded. If it is single-stranded, it can be either coding or non-coding (antisense).
  • the polynucleotide sequence encoding this mature polypeptide may be the same as the nucleic acid sequence shown in SEQ ID No. 1, or it may be the same mature polypeptide encoding the protein shown in SEQ ID No. 2, but shown in SEQ ID No. 1. Polynucleotides with different nucleic acid sequences.
  • the polynucleotide in the present invention includes: the coding sequence of the mature polypeptide only; the coding sequence of the mature polypeptide and various additional coding sequences; the coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.
  • polynucleotide encoding a polypeptide refers to a polynucleotide comprising a polynucleotide encoding the polypeptide and a polynucleotide comprising additional coding and / or non-coding sequences.
  • the invention also relates to the polynucleotide variants described above, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the invention.
  • This variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants. It can be seen from the above that an allelic variant is a replacement form of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change the function of the encoded polypeptide.
  • the polynucleotide of the present invention may also be: a polynucleotide comprising a leader sequence; a polynucleotide encoding a polypeptide (proteinogen) having a mature protein and additional 5 'amino acid residues.
  • the polynucleotide of the present invention can encode a mature protein, or a sequence protein, or a protein having both a sequence sequence and a leader sequence.
  • the present invention also relates to a polynucleotide that can hybridize to a polynucleotide that is 50% to 70% identical to the polynucleotide sequence described in the present invention.
  • the invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions.
  • strict conditions means: (1) at lower ionic strength Hybridization and elution at higher temperatures, such as 0.2xSSC, 0.1% SDS, 60C; or (2) adding a denaturant during hybridization, such as 50% (v / v) formamidine, 0.1 % Calf serum / 0.1% Ficoll, 42C, etc .; or (3) hybridization occurs only when the identity between the two sequences is 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 present invention provides an isolated human thromboprotein-1 mediating factor polypeptide, which is a polypeptide comprising the amino acid sequence of SEQ ID No. 2, or a conservative variant thereof, or an active fragment thereof, or Its active derivative.
  • a preferred polypeptide of the invention is a polypeptide comprising the amino acid sequence of SEQ ID No. 2.
  • the terms' fragment ',' derivative, and 'variant' refer to a polypeptide having the same biological function or activity as the polypeptide according to the present invention. These analogs also include proteolytic enzymes that can be activated to produce active mature polypeptides.
  • the polypeptide in 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 prokaryotic or eukaryotic host cells (for example, bacteria, yeast, higher plants, insects, and mammalian cells) using recombinant technology. Depending on the host cell used, the polypeptide of the present invention may be glycosylated or non-glycosylated.
  • the polypeptides of the invention may also include a starting methionine residue.
  • polypeptides and polynucleotides in the present invention are preferably provided in an isolated form, and purification uniformity is preferably achieved.
  • the present invention also relates to a vector comprising a polynucleotide of the present invention, a genetically engineered host cell containing the polynucleotide of the present invention, and a method for producing a polypeptide of the present invention by recombinant techniques.
  • Vectors that can be used in the present invention to contain polynucleotide sequences include chromosomal, non-chromosomal and synthetic DNA sequences, such as bacterial plasmids; phage DNA; fermentation Mother plasmid; plant cell virus; mammalian cell virus such as adenovirus, retrovirus.
  • any plasmid and vector can be used as long as it can replicate and survive in the host.
  • the expression vector preferably contains genes that provide the phenotypic traits of the selected transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescent protein (GFP), or for E. coli Tetracycline or ampicillin resistance.
  • genes that provide the phenotypic traits of the selected transformed host cells such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescent protein (GFP), or for E. coli Tetracycline or ampicillin resistance.
  • An appropriate expression control sequence can be ligated to the DNA sequence in the expression vector to direct mRNA synthesis.
  • promoters are: the lac or trp promoter of E. coli; the lambda phage PL promoter; eukaryotic promoters including CMV immediate early, HSV thymidine kinase, early and late SV40, 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 for translation initiation and a transcription terminator.
  • Enhancers are homeopathic factors of DNA, 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, cellular mast virus early promoter enhancers, polyoma enhancers on the late side of origin of replication, and adenoviral enhancers.
  • the host cell can be 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.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: E. coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; CH0, COS or Bowes melanoma cells Animal cells; adenovirus, etc.
  • the target polynucleotide sequence can be inserted into the appropriate restriction enzyme site on the vector through various steps.
  • the constructed vector can be used To transform the appropriate host cell to express this protein.
  • the selected promoter can be induced by appropriate methods (such as temperature conversion or chemical induction), and the cells can be cultured for a period of time.
  • the cells are harvested after centrifugation, and the cells are disrupted by physical or chemical methods, and the resulting crude extract is reserved for further purification.
  • Disruption includes freeze-thaw method, ultrasonic method, mechanical command fragmentation method, or use of cell lysis reagent.
  • Precipitation with ammonium sulfate or ethanol, acid extraction, anion or cation exchange chromatography, phosphate fiber chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography or phytohormone chromatography can be used
  • the protein was recovered and purified from the recombinant cell culture. If desired, a protein refolding step can be used to complete the conformation of the mature protein. Finally, high-performance liquid chromatography (HPLC) can be used to complete the final purification step.
  • HPLC high-performance liquid chromatography
  • Human thromboprotein-1 mediator regulates cell migration, cell proliferation, neurite growth, and angiogenesis through its adhesion to molecules in the cell, so human thromboprotein-1 mediates protein It can be used medically to treat diseases of the immune system, malignant tumors, etc., such as: acquired and inherited immunodeficiency, degenerative neurological diseases, spinal dysplasia, local anemia injuries, and osteosclerosis.
  • the polypeptides of the present invention are mainly used in the diagnosis and treatment of malignant tumors, including leukemias and lymphomas; tumors of epithelial cell origin; tumors of mesenchymal origin, such as sarcomas; central nervous system tumors, particularly preferably, they can be used for treatment Human malignant melanoma.
  • the polypeptide of the present invention is also used to treat chronic infectious diseases, including hepatitis, herpes, aphthous stomatitis, and AIDS.
  • the polypeptide of the present invention also has effects on damages, defects or disorders of immune tissues, and is particularly useful for diseases of the hematopoietic system (such as malignant anemia), skin diseases (such as squamous cell carcinoma), and autoimmune diseases (such as rheumatoid joints). Inflammation), and radiation sickness Wait.
  • diseases of the hematopoietic system such as malignant anemia
  • skin diseases such as squamous cell carcinoma
  • autoimmune diseases such as rheumatoid joints.
  • Inflammation and radiation sickness Wait.
  • the human thrombus-1 mediator of the present invention has 99% homology with the thrombus-1 mediator (Muskelin) of the squirrel, but the polypeptide of the present invention is derived from a human cDNA library and is more suitable for humans.
  • the disease treatment is highly specific and does not produce antibodies.
  • human thromboprotein-1 There are many ways to detect the expression of human thromboprotein-1, which provides a way to diagnose mutant or abnormal levels of human thromboprotein-1 expression. Therefore, the human thrombus-1 mediator and its antagonists can be used for the treatment of various diseases according to the diagnosis results, and the expression level of human thromboprotein-1 mediator can be monitored during the treatment of various diseases.
  • Antagonists of human thromboprotein-1 mediators include selected antibodies, compounds, receptor deletions, and the like.
  • Antagonists of human thromboprotein-1 mediators can bind to human thromboprotein-1 mediators and eliminate their functions, or inhibit the production of human thromboprotein-1 mediators, or with peptide activity Site binding prevents the polypeptide from performing its biological function.
  • Antagonists of the human thrombus-1 mediator can treat many tumor-related diseases, such as human malignant melanoma, human epithelial squamous cell carcinoma, and the like.
  • the polypeptide of the present invention can be used as an antigen to produce antibodies.
  • These antibodies can be polyclonal or monoclonal antibodies and are more suitable for human applications.
  • Techniques for preparing monoclonal antibodies include hybridoma technology, human B-cell hybridoma technology, and EBV-hybridoma technology.
  • the present invention also provides a method for selecting a human compound that mimics, promotes or inhibits the activity of thromboprotein-1 mediator, which comprises utilizing the polypeptide of the present invention. It also relates to a compound obtained by this method, which mimics, promotes or inhibits the activity of thrombin-1 mediation factor.
  • human thromboprotein-1 mediators When screening compounds as antagonists, human thromboprotein-1 mediators can be added to bioanalytical assays to influence human thromboprotein-1 mediators by measuring compounds The interaction between a factor and its receptor determines whether a compound is an antagonist.
  • the present invention Based on the biological activity of the polypeptide, the polynucleotide of the present invention, and the compound obtained by the screening method of the present invention, the present invention also provides the above-mentioned substance in the preparation of a medicament for treating a disease associated with abnormal activity of a human thromboembolic protein-1 mediating factor.
  • these pharmaceutical compositions can be used to treat or prevent acquired and hereditary immunodeficiency, degenerative neurological diseases, spinal cord dysplasia, local anemia injuries, osteosclerosis, malignant tumors or chronic infectious diseases.
  • the polypeptides and antagonists of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffered salts, glycerol, and combinations thereof.
  • the composition contains an effective amount of a 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 polynucleotide of the present invention can be used for the treatment of a disease, that is, gene therapy. These include cell in vivo engineering and antisense constructs.
  • a patient is provided with a production cell that produces a viral particle containing RNA encoding a polypeptide of the present invention to engineer the cell in vivo and express the polypeptide in vivo.
  • the expression vector of the engineered cell may be a retrovirus, an adenovirus, or the like.
  • antisense constructs can be accomplished by using antisense DNA and RNA to control gene expression.
  • the 5 'coding region of a polynucleotide sequence encoding a mature polypeptide of the present invention is used to design an antisense RNA oligonucleotide having a length of from 10 to 40 base pairs.
  • Antisense RNA oligonucleotides hybridize with mRNA in the body and block translation of the mRNA molecule by adult thromboprotein-1 mediators, or antisense RNA oligonucleotides are delivered into cells to make antisense RNA or DNA Expressed in vivo, thereby inhibiting the production of human thromboprotein-1 mediated factors.
  • the polynucleotide of the present invention can also be used for diagnosis. Such as detection at the chromosome level, detection at the DNA level, and so on. Fluorescent in situ hybridization (FISH) of cDNA clones onto metaphase chromosome bands can provide precise one-step chromosomal localization. When the sequence is accurately located on the chromosome, the physical position of the sequence on the chromosome can be matched with the genetic map data. Then, through analysis, the relationship between the polynucleotide of the present invention and some diseases that have been located on the chromosome can be determined.
  • FISH fluorescent in situ hybridization
  • the full-length human thrombus-1 mediator factor gene and fragments thereof can be used to detect a human thrombus-1 mediator-related disease or susceptibility diagnosis.
  • Nucleic acids obtained from a patient's blood, spinal cord, and tissue anatomy can be used to detect mutations at the DNA level by various techniques. Such as hybridization, chemical lysis, direct sequencing of DNA, Southern hybridization of genomic DNA, etc. Examples
  • Total human fetal brain RNA was extracted by one-step method with guanidine isothiocyanate / pan / chloroform.
  • Oly (A) mRNA was isolated from total RNA using Quik mRNA Isolation Kit (Qiagene). 2ug poly (A) mRNA was formed into cDNA by reverse transcription. The Smart cDNA cloning kit (purchased from Clontech) was used to insert the cDNA fragment into the multiple cloning site of the body to transform the DH5a bacteria to form a cDNA library. A total of 3028 clones were obtained. The dideoxy method was used to determine the sequence of the 5, and 3, ends of all clones.
  • the determined cDNA sequence was compared with the existing public DNA sequence database, and it was found that a cloned DNA sequence was new DNA with a gene length of 3011bp.
  • a series of primers were synthesized to determine the DNA sequence of the clone in both directions.
  • Computer analysis showed that the full-length cDNA contained in the clone was a new DNA sequence (as shown in Seq ID No 2), with a 2207bp ORF from 24bp to 2231bp, encoding a new protein (such as Seq ID No 1 As shown).
  • Seq ID No 2 a new DNA sequence
  • a 2207bp ORF from 24bp to 2231bp
  • Example 2 Expression and purification of a novel human thromboprotein-1 mediator in E. coli
  • Primer 2 5 'GTTTAGCGGGATACCCTCAGG 3'
  • the two primer sequences contain Apal and EcoRl obscure sites, respectively, followed by the coding sequences of the target gene 3, 5 and 5, respectively, and a PCR reaction is performed using the PBS plasmid containing the full-length target gene as a template.
  • the restriction sites of Apal and EcoR1 correspond to the selective endonuclease sites on the expression vector plasmid PTSA-18.
  • Apal and EcoRl were used to cut and ligate the amplified sequence and plasmid PTSA-18, respectively.
  • the recombinant plasmid was transformed into the host strain E. coli BL21 (DE3) plySs and induced by IPTG for expression.
  • the expression product was subjected to ultrasonic lysis and thermal denaturation, and then applied to a DEAE column to obtain a purified target human thromboembol-1 mediation factor protein.
  • Example 3 Homologous search of cDNA clones
  • the sequence of the human thromboembolic protein-1 mediation factor polynucleotide provided by the present invention and its encoded protein sequence were searched in databases such as Genbank, Swiss sport and other databases.
  • the program used for the search was Blast (Basic local Alignment search tool) (1993 Proc Nat Acad Sci 90: 5873-5877), Blast can find many genes that are homologous to the human thrombus-1 mediator, among which the gene with the most homology to the gene we invented, which encodes
  • the accession number of the protein in Genbank is u72194. These retrieved gene or protein sequences can be retrieved from the Genbank database.
  • the recalled sequence can be compared with the Pileup (multi-sequence) and Gap (two-sequence) programs in the GCG software package. Functional prediction of new proteins can Analysis was performed using the Motif program. The results show that the human thrombus-1 mediator provided by the present invention has 99% homology with the thrombus-1 mediator (Muskelin) of the squirrel.
  • This method involves acid guanidinium thiocyanate-chloroform extraction. That is, the tissue is homogenized with 4M guanidine isothiocyanate-25mM sodium citrate, 0.2M sodium acetate (pH4.0), and 1 volume of phenol and 1/5 volume of chloroform-isoamyl alcohol (49 : 1), centrifuge after mixing. Aspirate the aqueous layer, add isopropanol (0.8 vol) and centrifuge the mixture to obtain RNA precipitate.
  • RNA precipitate was washed with 70% ethanol, dried and dissolved in water.
  • electrophoresis was performed on a 1.2% agarose gel containing 20 mM 3- (N-morpholino) propanesulfonic acid (pH 7.0)-5 mM sodium acetate-1 mM EDTA-2. 2M formaldehyde. It was then transferred to a nitrocellulose membrane.
  • 32P-labeled probe (approximately 2x10 cpm / ml) in a solution After hybridization at 42 overnight, the solution contained 50% formamide-25mM KH2P04 (pH7.4)-5xSSC-5xDenhardt's solution and 200 g / ml salmon sperm DNA.
  • a 32P-labeled DNA probe was prepared by a random primer method using ⁇ -32P dATP. The DNA probe used for PCR amplified the sequence of the human thrombus-1 mediating factor coding region. After hybridization, the filter was washed in lxSSC-0. 1% SDS at 65 "for 30 minutes. Then, it was analyzed and quantified using a Phosphor Imager. The results showed that the human thrombus-1 mediator gene can be specifically expressed in spinal cord tissue
  • Example 5 Preparation of antibodies against human thromboprotein-1 mediator:
  • Polypeptide synthesizer (PE-ABI) was used to synthesize the following human thrombin-1 mediator factor thrombin-1 mediator-specific polypeptide: Lys Leu Glu Arg Pro Ala lie Val Gin Asn (amino acid sequence 61- 70-bit).
  • the polypeptide is coupled to hemocyanin and bovine serum albumin to form a complex, respectively.
  • Rabbits were immunized with mg of the hemocyanin-polypeptide complex plus complete Freund's adjuvant, and 15 days later the blood-blue protein peptide complex plus incomplete Freund's adjuvant was used to boost immunity once.
  • a titer plate coated with a 15 g / nil bovine serum albumin peptide complex was used as an ELISA to determine antibody titers in rabbit serum.
  • Protein A-Sepharose was used to isolate total IgG from antibody-positive rabbit serum.
  • the peptide was bound to a cyanogen bromide-activated Sepharose 4B column and the anti-peptide antibody was isolated from the total IgG by affinity chromatography.
  • the immunoprecipitation method demonstrated that the purified antibody can specifically bind to the human thromboprotein-1 mediating factor of the present invention.

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Abstract

L'invention concerne un nouveau type de muskeline et de polynucléotides codant pour des polypeptides, ainsi qu'un procédé de production de ces polypeptides par des méthodes de recombinaison. Elle concerne également une méthode d'application de ces polypeptides pour le traitement de nombreux types de maladies, telles que le syndrome d'immunodéficience acquis, la neuropathie rétrograde, la déficience du développement spinal, les lésions anémiques topiques et l'ostéopathie. L'invention concerne en outre un anticorps et un antagoniste de ce polypeptide, ainsi que leur utilisation thérapeutique. Par ailleurs, elle se rapporte à l'utilisation de polynucléotides codant pour ces protéines.
PCT/CN2000/000269 1999-09-10 2000-09-11 Nouvelle muskeline et ses procedes d'application et de production WO2001019865A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN00812875A CN1387535A (zh) 1999-09-10 2000-09-11 一种新的人凝血栓蛋白－1介导因子及其应用和制备方法

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CN99116856.9 1999-09-10
CN99116856A CN1288056A (zh) 1999-09-10 1999-09-10 一种新的人凝血栓蛋白-1介导因子及其应用和制备方法

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
EMBO J., vol. 17, no. 17, 1998, pages 4964 - 4974 *

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