WO2023097475A1 - Procédé de construction d'un vecteur d'expression eucaryote de protéine pak1 humaine, procédé d'expression et procédé de purification - Google Patents

Procédé de construction d'un vecteur d'expression eucaryote de protéine pak1 humaine, procédé d'expression et procédé de purification Download PDF

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Publication number
WO2023097475A1
WO2023097475A1 PCT/CN2021/134500 CN2021134500W WO2023097475A1 WO 2023097475 A1 WO2023097475 A1 WO 2023097475A1 CN 2021134500 W CN2021134500 W CN 2021134500W WO 2023097475 A1 WO2023097475 A1 WO 2023097475A1
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WIPO (PCT)
Prior art keywords
pak1
cells
protein
expression vector
eukaryotic expression
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PCT/CN2021/134500
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English (en)
Chinese (zh)
Inventor
张浩洋
石坚
徐爱华
朱国方
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绍兴守仁医疗健康科技有限公司
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Priority to PCT/CN2021/134500 priority Critical patent/WO2023097475A1/fr
Publication of WO2023097475A1 publication Critical patent/WO2023097475A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)

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  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Procédé de construction d'un vecteur d'expression eucaryote d'une protéine PAK1 humaine, comprenant les étapes suivantes : 1) amplification par PCR pour obtenir un fragment de gène PAK1 ; 2) liaison à un vecteur d'expression eucaryote pour obtenir un plasmide recombiné ; et 3) transfection, transformation et réplication pour obtenir un vecteur d'expression eucaryote d'une protéine PAK1 humaine. Est également divulgué un procédé d'expression d'une protéine PAK1 humaine recombinée, comprenant en outre l'étape 4) : transfection d'un plasmide du vecteur d'expression eucaryote de la protéine PAK1 humaine obtenu à l'étape 3) dans des cellules eucaryotes en vue de l'expression. Est également divulgué un procédé d'obtention d'une protéine PAK1 humaine recombinée, comprenant en outre l'étape 5) : mise en culture du clone positif obtenu à l'étape 4) pour accroître un système, collecte des cellules cultivées, réalisation d'une centrifugation pour collecter le surnageant, et réalisation d'une purification au moyen d'une colonne chromatographique pour obtenir une protéine PAK1 humaine recombinée.
PCT/CN2021/134500 2021-11-30 2021-11-30 Procédé de construction d'un vecteur d'expression eucaryote de protéine pak1 humaine, procédé d'expression et procédé de purification WO2023097475A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/134500 WO2023097475A1 (fr) 2021-11-30 2021-11-30 Procédé de construction d'un vecteur d'expression eucaryote de protéine pak1 humaine, procédé d'expression et procédé de purification

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/134500 WO2023097475A1 (fr) 2021-11-30 2021-11-30 Procédé de construction d'un vecteur d'expression eucaryote de protéine pak1 humaine, procédé d'expression et procédé de purification

Publications (1)

Publication Number Publication Date
WO2023097475A1 true WO2023097475A1 (fr) 2023-06-08

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PCT/CN2021/134500 WO2023097475A1 (fr) 2021-11-30 2021-11-30 Procédé de construction d'un vecteur d'expression eucaryote de protéine pak1 humaine, procédé d'expression et procédé de purification

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WO (1) WO2023097475A1 (fr)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7005285B1 (en) * 1999-11-15 2006-02-28 Pharmacia & Italia S.P.A. Human p21-activated kinase 5 polypeptide
CN1826130A (zh) * 2003-07-18 2006-08-30 塞诺菲-安万特德国有限公司 Pak抑制物用于治疗关节疾病的用途
CN101778641A (zh) * 2007-06-19 2010-07-14 武田药品工业株式会社 癌症的预防或治疗剂
WO2014150790A1 (fr) * 2013-03-15 2014-09-25 Albert Einstein College Of Medicine Of Yeshiva University Inhibition de pak1 pour traiter une leucémie aiguë myéloïde et les syndromes myélodysplasiques
CN111808833A (zh) * 2020-07-21 2020-10-23 南通大学 Cdc42蛋白片段的构建及其抗胰腺癌侵袭的活性应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7005285B1 (en) * 1999-11-15 2006-02-28 Pharmacia & Italia S.P.A. Human p21-activated kinase 5 polypeptide
CN1826130A (zh) * 2003-07-18 2006-08-30 塞诺菲-安万特德国有限公司 Pak抑制物用于治疗关节疾病的用途
CN101778641A (zh) * 2007-06-19 2010-07-14 武田药品工业株式会社 癌症的预防或治疗剂
WO2014150790A1 (fr) * 2013-03-15 2014-09-25 Albert Einstein College Of Medicine Of Yeshiva University Inhibition de pak1 pour traiter une leucémie aiguë myéloïde et les syndromes myélodysplasiques
CN111808833A (zh) * 2020-07-21 2020-10-23 南通大学 Cdc42蛋白片段的构建及其抗胰腺癌侵袭的活性应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Nucleotide 21 November 2020 (2020-11-21), ANONYMOUS : "PREDICTED: Homo sapiens p21 (RAC1) activated kinase 1 (PAK1), transcri -Nucleotide -NCBI", XP093069655, retrieved from NCBI Database accession no. XM_024448560 *
WU, JINBAO ET AL.: "Construction of a Eukaryotic Green Fluorescent Protein Expression Vector Carrying the PAK-1 Gene (pEGFP-C1/PAK1) and its Expression in SW480 Cells", WORLD CHINESE JOURNAL OF DIGESTOLOGY, SHIJE WEI-CHANGBINGXUE ZAZHISHE , TAIYUAN, CN, vol. 19, no. 26, 18 September 2011 (2011-09-18), CN , pages 2730 - 2734, XP009546743, ISSN: 1009-3079, DOI: 10.11569/wcjd.v19.i26.2730 *

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