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WO2001096560A1 - Nouveau polypeptide, proteine humaine stat2, et polynucleotide codant ce polypeptide - Google Patents

Nouveau polypeptide, proteine humaine stat2, et polynucleotide codant ce polypeptide Download PDF

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Publication number
WO2001096560A1
WO2001096560A1 PCT/CN2001/000718 CN0100718W WO0196560A1 WO 2001096560 A1 WO2001096560 A1 WO 2001096560A1 CN 0100718 W CN0100718 W CN 0100718W WO 0196560 A1 WO0196560 A1 WO 0196560A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
protein
human
sequence
Prior art date
Application number
PCT/CN2001/000718
Other languages
English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
Original Assignee
Shanghai Biowindow Gene Development Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shanghai Biowindow Gene Development Inc. filed Critical Shanghai Biowindow Gene Development Inc.
Priority to AU87481/01A priority Critical patent/AU8748101A/en
Publication of WO2001096560A1 publication Critical patent/WO2001096560A1/fr

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Classifications

    • 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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity

Definitions

  • An object of the present invention is to provide an isolated novel polypeptide-human STAT2 protein 10 and fragments, analogs and derivatives thereof.
  • Another object of the invention is to provide a polynucleotide encoding the polypeptide.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors directed to the polypeptide-human STAT2 protein 10 of the present invention.
  • the present invention relates to an isolated polypeptide.
  • the polypeptide is of human origin and comprises: a polypeptide having the amino acid sequence of SEQ ID No. 2, or a conservative variant, biologically active fragment or derivative thereof.
  • the polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 2.
  • the invention also relates to an isolated polynucleotide comprising a nucleotide sequence or a variant thereof selected from the group consisting of:
  • the invention also relates to a method for in vitro detection of a disease or susceptibility to disease associated with abnormal expression of human STAT2 protein 10 protein, which comprises detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or detecting a mutation in a biological sample.
  • the amount or biological activity of a polypeptide of the invention comprises detecting a mutation in the polypeptide or a polynucleotide sequence encoding the same in a biological sample, or detecting a mutation in a biological sample.
  • a polynucleotide sequence encoding the human STAT2 protein 10 may be inserted into a vector to form a recombinant vector containing the polynucleotide of the present invention.
  • vector refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses or other vectors well known in the art.
  • Vectors suitable for use in the present invention include, but are not limited to: T7 promoter-based expression vectors expressed in bacteria (Rosenberg, et al.
  • coli Streptomyces
  • bacterial cells such as Salmonella typhimurium
  • fungal cells such as yeast
  • plant cells such as insect cells such as fly S2 or Sf9
  • animal cells such as CH0, COS, or Bowes melanoma cells.
  • Transformation of a host cell with a DM sequence according to the present invention or a recombinant vector containing the DM sequence can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DM can be harvested after the exponential growth phase and treated with CaCl. The steps used are well known in the art. Alternatively, MgCl 2 is used. If necessary, transformation can also be performed by electroporation.
  • the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, or conventional mechanical methods such as microinjection, electroporation, and liposome packaging.
  • Antagonists of human STAT2 protein 10 include antibodies, compounds, receptor deletions, and analogs. Antagonists of human STAT2 protein 10 can bind to human STAT2 protein 10 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.
  • the present invention provides a method for producing antibodies using polypeptides, and fragments, derivatives, analogs or cells thereof as antigens. These antibodies can be polyclonal or monoclonal antibodies.
  • the invention also provides antibodies against the human STAT2 protein 10 epitope. These antibodies include (but are not limited to): Doklon antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, and fragments produced by Fab expression libraries.
  • Oligonucleotides including antisense RM and DNA
  • ribozymes that inhibit human STAT2 protein 10 raRNA are also within the scope of the present invention.
  • a ribozyme is an enzyme-like RNA molecule that specifically decomposes specific RNA. Its mechanism of action is that the ribozyme molecule specifically hybridizes with a complementary target MA for endonucleation.
  • Antisense RNA, DM, and ribozymes can be obtained using any existing RNA or DM synthesis techniques, such as solid-phase phosphate amide chemical synthesis to synthesize oligonucleotides.
  • Antisense RM molecules can be obtained by in vitro or in vivo transcription of a DNA sequence encoding the RNA.
  • PCR primers (preferably 15-35bp) are prepared based on cDM, and the sequences can be located on chromosomes. These primers were then used for PCR screening of somatic hybrid cells containing individual human chromosomes. Only those heterozygous cells containing the human gene corresponding to the primer will produce amplified fragments.
  • Fluorescent in situ hybridization of cDNA clones with metaphase chromosomes allows precise chromosomal localization in one step.
  • FISH Fluorescent in situ hybridization
  • the polypeptides, polynucleotides and mimetics, agonists, antagonists and inhibitors of the present invention can be used in combination with a suitable pharmaceutical carrier.
  • suitable pharmaceutical carrier can be water, glucose, ethanol, salts, buffers, glycerol, and combinations thereof.
  • the composition comprises a safe and effective amount of the polypeptide or antagonist, and carriers and excipients which do not affect the effect of the drug. These compositions can be used as drugs for the treatment of diseases.
  • Primer 1 5,-GCTGGTGGGAAGGGAATAAAGAAG -3, (SEQ ID NO: 3)
  • Primer 2 5,-GCAGAGGAAATTCAGGAATTCACG -3, (SEQ ID NO: 4)
  • Primerl is a forward sequence located at the 5th end of SEQ ID NO: 1, starting at lbp; Pr imer2 is the 3′-end reverse sequence in SEQ ID NO: 1.
  • RNA probe was the PCR amplified human STAT2 protein 10 coding region sequence (1379bp to 1651bp) shown in FIG. 1.
  • Pr imer 3 5'- CCCCATATGATGGGGACCCTCCAGGGCAAGGGC -3, (Seq ID No: 5)
  • Primer4 5'- CATGGATCCTCATGGGGCCCTTGGGCAGCAGGG -3, (Seq ID No: 6)
  • the 5 'ends of these two primers contain Ndel and BamHI digestion sites, respectively Points, followed by the coding sequences of the 5 'and 3' ends of the gene of interest, respectively.
  • the Ndel and BamHI restriction sites correspond to the expression vector plasmid pET-28b (+) (Novagen, Cat. No. 69865. 3) Selective endonuclease site.
  • the ligation product was transformed into coliform bacteria DH5a by the calcium chloride method, and cultured overnight on LB plates containing kanamycin (final concentration 3 ( ⁇ g / ml)), and positive clones were selected by colony PCR method and sequenced.
  • a positive clone with the correct sequence (pET-0452a05) was used to transform the recombinant plasmid into E. coli BL21 (DE3) plyS S (N OV agen) by calcium chloride method.
  • the purpose of this embodiment is to select a suitable oligonucleotide fragment from the polynucleotide SEQ ID NO: 1 of the present invention as a hybridization probe, and to identify whether some tissues contain the polynucleoside of the present invention by using a filter hybridization method.
  • Filter hybridization methods include dot blotting, Southern blotting, Northern blotting, and copying methods. They all use the same steps of hybridization after fixing the polynucleotide sample to be tested on the filter.
  • step 8-13 are only used when contamination must be removed, otherwise step 14 can be performed directly.
  • Gene chip or gene microarray is a new technology currently being developed by many national laboratories and large pharmaceutical companies. It refers to the orderly and high-density arrangement of a large number of target gene fragments on glass, The data is compared and analyzed on a carrier such as silicon using fluorescence detection and computer software to achieve the purpose of rapid, efficient, and high-throughput analysis of biological information.
  • the polynucleotide of the present invention can be used as target DNA for gene chip technology for high-throughput research on the function of new genes; search for and screen new tissue-specific genes, especially diseases related genes such as tumors; diagnosis of diseases such as heredity disease.
  • Total mRNA was extracted from human mixed tissues and specific tissues (or stimulated cell lines) in one step, and the mRNA was purified with Ol igotex mRNA Midi Ki t (purchased from QiaGen).
  • JY Cy3dUTP (5-Amino-propargyl-2'-deoxyuridine 5--triphate coupled to Cy3 f luorescent dye, purchased from Amersham Phamacia Biotech) was used to label the mRNA of human mixed tissue, and the fluorescent reagent Cy5dUTP (5-Amino- propargyl -2'-deoxyuridine 5'-triphate coupled to Cy5 fluorescent dye, purchased from Amersham Phamacia Biotech Company, labeled mRM of specific tissues (or stimulated cell lines) of the body, and the probes were prepared after purification.
  • Cy5dUTP (5-Amino- propargyl -2'-deoxyuridine 5'-triphate coupled to Cy5 fluorescent dye, purchased from Amersham Pham

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un nouveau polypeptide, une protéine humaine STAT2, et un polynucléotide codant ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, des troubles du développement, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant la protéine humaine STAT2.
PCT/CN2001/000718 2000-05-09 2001-05-08 Nouveau polypeptide, proteine humaine stat2, et polynucleotide codant ce polypeptide WO2001096560A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU87481/01A AU8748101A (en) 2000-05-09 2001-05-08 A novel polypeptide, a human protein stat2 and the polynucleotide encoding the polypeptide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN 00115612 CN1322737A (zh) 2000-05-09 2000-05-09 一种新的多肽——人Stat2蛋白10和编码这种多肽的多核苷酸
CN00115612.8 2000-05-09

Publications (1)

Publication Number Publication Date
WO2001096560A1 true WO2001096560A1 (fr) 2001-12-20

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PCT/CN2001/000718 WO2001096560A1 (fr) 2000-05-09 2001-05-08 Nouveau polypeptide, proteine humaine stat2, et polynucleotide codant ce polypeptide

Country Status (3)

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CN (1) CN1322737A (fr)
AU (1) AU8748101A (fr)
WO (1) WO2001096560A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005075508A2 (fr) * 2004-02-10 2005-08-18 Forschungsverbund Berlin E.V. Molecules stat hyperactives et leur utilisation dans des dosages dans lesquels l'activation genique est utilisee
EP1637540A1 (fr) * 2004-09-17 2006-03-22 Forschungsverbund Berlin e.V. Des molécules hyperactives de stat et procédés d'essai de l'activation génique

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 21 December 1999 (1999-12-21), Database accession no. AC006317 *
DATABASE GENBANK [online] 23 November 1999 (1999-11-23), accession no. EMBL Database accession no. AL035400.13 *
DATABASE GENBANK [online] 3 July 1998 (1998-07-03), Database accession no. AC004552 *
DATABASE GENBANK [online] 4 February 2000 (2000-02-04), Database accession no. AC002530 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005075508A2 (fr) * 2004-02-10 2005-08-18 Forschungsverbund Berlin E.V. Molecules stat hyperactives et leur utilisation dans des dosages dans lesquels l'activation genique est utilisee
WO2005075508A3 (fr) * 2004-02-10 2006-07-20 Forschungsverbund Berlin Ev Molecules stat hyperactives et leur utilisation dans des dosages dans lesquels l'activation genique est utilisee
EP1637540A1 (fr) * 2004-09-17 2006-03-22 Forschungsverbund Berlin e.V. Des molécules hyperactives de stat et procédés d'essai de l'activation génique

Also Published As

Publication number Publication date
AU8748101A (en) 2001-12-24
CN1322737A (zh) 2001-11-21

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