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WO2001047969A1 - Nouveau polypeptide, proteine de translocation nucleaire i10, et polynucleotide codant pour ce polypeptide - Google Patents

Nouveau polypeptide, proteine de translocation nucleaire i10, et polynucleotide codant pour ce polypeptide Download PDF

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Publication number
WO2001047969A1
WO2001047969A1 PCT/CN2000/000598 CN0000598W WO0147969A1 WO 2001047969 A1 WO2001047969 A1 WO 2001047969A1 CN 0000598 W CN0000598 W CN 0000598W WO 0147969 A1 WO0147969 A1 WO 0147969A1
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WO
WIPO (PCT)
Prior art keywords
polypeptide
polynucleotide
protein
sequence
seq
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CN2000/000598
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English (en)
Chinese (zh)
Inventor
Yumin Mao
Yi Xie
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai BioDoor Gene Technology Ltd
Fudan University
Original Assignee
Shanghai BioDoor Gene Technology Ltd
Fudan University
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 BioDoor Gene Technology Ltd, Fudan University filed Critical Shanghai BioDoor Gene Technology Ltd
Priority to AU21414/01A priority Critical patent/AU2141401A/en
Publication of WO2001047969A1 publication Critical patent/WO2001047969A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Another object of the present invention is to provide an antibody against the polypeptide-nuclear conversion protein ⁇ 0 of the present invention.
  • Another object of the present invention is to provide mimetic compounds, antagonists, agonists, and inhibitors directed to the polypeptide-nuclear conversion protein no of the present invention.
  • 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.
  • “Deletion” refers to the deletion of one or more amino acids or nucleotides in an amino acid sequence or nucleotide sequence.
  • 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 separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state. .
  • "strict conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2xSSC, 0.1% SDS, 6 (TC; or (2) Add denaturants during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Fi col 1, 42 ° C, etc .; or (3) only between two sequences Hybridization occurs only when the identity is at least 95%, and more preferably 97%, and the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2 .
  • 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 nuclear conversion protein ⁇ 0 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) expression libraries Antibody screening to detect cloned polynucleotide fragments with common structural characteristics.
  • 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 transcript of the nuclear transfer protein ⁇ 0; (4) the Immunological techniques or assays for biological activity to detect gene-expressed protein products. The above methods can be used singly or in combination.
  • Methods well known to those skilled in the art can be used to construct an expression vector containing a DNA sequence encoding nuclear conversion protein 110 and appropriate transcriptional / translational regulatory elements. These methods include in vitro recombinant DM technology, DNA synthesis technology, and in vivo recombination technology (Sambroook, et al. Molecular Cloning, a Laboratory Manul, Cold Spring Harbor Laboratory. New York, 1989).
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. Representative examples of these promoters are: the lac or trp promoter of E.
  • E. 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.
  • the polynucleotide sequence of the present invention can be used to express or produce a recombinant nuclear transfer protein I 1 0 (Scence, 1984; 224: 1431). Generally there are the following steps:
  • the invention also provides methods for screening compounds to identify agents that increase (agonist) or suppress (antagonist) nuclear transduction protein 110.
  • Agonists enhance nuclear conversion proteins ⁇ 0 to stimulate biological functions such as cell proliferation, while antagonists prevent and treat disorders related to excessive cell proliferation, such as various cancers.
  • mammalian cells or membrane preparations expressing nuclear conversion protein ⁇ 0 can be cultured together with labeled nuclear conversion protein 110 in the presence of drugs. The ability of the drug to increase or block this interaction is then determined.
  • Polyclonal antibodies can be produced by injecting nucleoprotein 110 directly into immunized animals (such as rabbits, mice, rats, etc.).
  • immunized animals such as rabbits, mice, rats, etc.
  • a variety of adjuvants can be used to enhance the immune response, including but not limited to Freund's adjuvant.
  • Techniques for preparing monoclonal antibodies to nuclear transfer protein 110 include, but are not limited to, hybridoma technology (Kohler and Milstein. Nature, 1975, 256: 495-497), triple tumor technology, human beta-cell hybridoma technology, and EBV-hybridoma technology. Technology, etc. Chimeric antibodies that bind human constant regions and non-human variable regions can be produced using existing techniques (Morrison et al, PNAS, 1985, 81: 6851). The existing technology for producing single-chain antibodies (U.S. Pat No. 4946778) can also be used to produce single-chain antibodies against nuclear conversion protein 110.
  • Nucleoconvertin 110 mutations include point mutations, translocations, deletions, recombinations, and any other abnormalities compared to the normal wild-type nucleotransin 110 DM 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.
  • the ligation product was transformed into E. coli DH5C by the calcium chloride method. After being cultured overnight on LB plates containing kanamycin (final concentration 30 ⁇ ⁇ / ⁇ 1), positive clones were selected by colony PCR method and sequenced. A positive clone (pET-0614hlO) with the correct sequence was selected, and the recombinant plasmid was transformed into E. coli BL21 (DE3) plySs (product of Novagen) by the calcium chloride method.
  • Polypeptide synthesizer (product of PE company) was used to synthesize the following nucleoprotein 110-specific peptides: NH 2 -Met-Va l-Glu-Ser-Thr-Ala-Met-Ala-Glu-Va l-Ser-Gly- Hi s-Arg-Gly-COOH (SEQ ID NO: 7).
  • the polypeptide was coupled to hemocyanin and bovine serum albumin to form a complex, respectively. For methods, see: Avrameas, et al. Immunochemi s try, 1969; 6: 43.
  • a total of 4,000 polynucleotide sequences of various full-length cDNAs are used as target DNA, including the polynucleotide of the present invention. They were amplified by PCR respectively. After purification, the concentration of the amplified product was adjusted to about 500 ng / ul, and spotted on a glass medium using a Cartesian 7500 spotter (purchased from Cartesian Company, USA). The distance between the points is 280 ⁇ . The spotted slides were hydrated and dried, cross-linked in a UV cross-linker, and dried after elution to fix the DNA on the glass slides to prepare chips. The specific method steps have been reported in the literature. The sample post-processing steps in this embodiment are:

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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)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un nouveau polypeptide, une protéine de translocation nucléaire I10, et un polynucléotide codant pour ce polypeptide ainsi qu'un procédé d'obtention de ce polypeptide par des techniques recombinantes d'ADN. L'invention concerne en outre les applications de ce polypeptide dans le traitement de maladies, notamment des tumeurs malignes, de l'hémopathie, de l'infection par VIH, de maladies immunitaires et de diverses inflammations. L'invention concerne aussi l'antagoniste agissant contre le polypeptide et son action thérapeutique ainsi que les applications de ce polynucléotide codant pour la protéine de translocation nucléaire I10.
PCT/CN2000/000598 1999-12-23 2000-12-18 Nouveau polypeptide, proteine de translocation nucleaire i10, et polynucleotide codant pour ce polypeptide Ceased WO2001047969A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU21414/01A AU2141401A (en) 1999-12-23 2000-12-18 A novel polypeptide-nuclear translocation protein i10 and the polynucleotide encoding said polypeptide

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN 99125724 CN1300764A (zh) 1999-12-23 1999-12-23 一种新的多肽－核转换蛋白i10和编码这种多肽的多核苷酸
CN99125724.3 1999-12-23

Publications (1)

Publication Number Publication Date
WO2001047969A1 true WO2001047969A1 (fr) 2001-07-05

Family

ID=5284135

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2000/000598 Ceased WO2001047969A1 (fr) 1999-12-23 2000-12-18 Nouveau polypeptide, proteine de translocation nucleaire i10, et polynucleotide codant pour ce polypeptide

Country Status (3)

Country Link
CN (1) CN1300764A (fr)
AU (1) AU2141401A (fr)
WO (1) WO2001047969A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996029406A2 (fr) * 1995-03-20 1996-09-26 The Rockefeller University Facteur de localisation nucleaire associe aux rythmes circadiens
WO1999003489A2 (fr) * 1997-07-21 1999-01-28 Ciblex Corporation Procedes et compositions permettant de reguler le transport des proteines dans le noyau

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996029406A2 (fr) * 1995-03-20 1996-09-26 The Rockefeller University Facteur de localisation nucleaire associe aux rythmes circadiens
WO1999003489A2 (fr) * 1997-07-21 1999-01-28 Ciblex Corporation Procedes et compositions permettant de reguler le transport des proteines dans le noyau

Also Published As

Publication number Publication date
AU2141401A (en) 2001-07-09
CN1300764A (zh) 2001-06-27

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