KR970006499A - Method for preparing erythropoietin - Google Patents

Method for preparing erythropoietin Download PDF

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Publication number
KR970006499A
KR970006499A KR1019950022441A KR19950022441A KR970006499A KR 970006499 A KR970006499 A KR 970006499A KR 1019950022441 A KR1019950022441 A KR 1019950022441A KR 19950022441 A KR19950022441 A KR 19950022441A KR 970006499 A KR970006499 A KR 970006499A
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dhfr
transformant
human erythropoietin
kctc
ecl
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KR1019950022441A
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Korean (ko)
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KR0162021B1 (en
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홍효정
김윤규
류춘제
김상직
박홍록
이상철
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김은영
한국과학기술연구원
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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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/505—Erythropoietin [EPO]
    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
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  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

본 발명은 플라스미드 pCMV-dhfr 및 이것을 이용하여 에리트로포이에틴(Erythropoietin;이하 EPO라고 약칭한다)을 제조하는 방법에 관한 것이다.The present invention relates to a plasmid pCMV-dhfr and a method for producing erythropoietin (hereinafter abbreviated as EPO) using the same.

본 발명은 특히 dhfr 유전자가 연결되어 있는 SV40 프로모터 128-270 부위를 제거하여 pRc/CMV에 삽입하여 발현벡타 pCMV-dhfr을 만들고 여기에 인간 EPO 유전자를 클로닝하여 pEpoG-dhfr 또는 pEpoC-dhfr을 만든후 이것으로 CHO 세포를 형질전환시켜 얻은 세포주 g2 또는 ECl으로부터 2nM 이하의 낮은 농도의 MTX 함유 α-MEM 배지에서 고역가의 EPO를 경제적으로 제조하는 방법에 관한 것이다.In particular, the present invention removes the 128-270 region of the SV40 promoter to which the dhfr gene is linked, inserts it into pRc / CMV to make expression vector pCMV-dhfr, and clones the human EPO gene to make pEpoG-dhfr or pEpoC-dhfr. This relates to a method for economically producing high titers of EPO in a low concentration of MTX-containing α-MEM medium of 2 nM or less from cell lines g2 or ECl obtained by transforming CHO cells.

Description

에리트로포이에틴의 제조방법Method for preparing erythropoietin

본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음Since this is an open matter, no full text was included.

Claims (13)

SV40 프로모터 128-270 부위를 제거하여 만든 프로모터에 연결시킨 dhfr 유전자를 함유하며 CMV 프로모터에 다클로닝부위(multi cloning site)를 연결시킨 벡터 pCMV-dhfr(KCTC 8671P).Vector pCMV-dhfr (KCTC 8671P) containing a dhfr gene linked to a promoter made by removing the SV40 promoter 128-270 and linking a multi cloning site to a CMV promoter. 제1항의 벡터 pCMV-dhfr에 유전자형 EPO 유전자를 삽입시켜 얻은 pEpoG-dhfr 플라스미드(KCTC 0181BP).PEpoG-dhfr plasmid obtained by inserting the genotype EPO gene into the vector pCMV-dhfr of claim 1 (KCTC 0181BP). 제1항의 벡터 pCMV-dhfr에 cDNA형 EPO 유전자를 삽입시켜 얻은 pEpoC-dhfr 플라스미드(KCTC 0180BP).PEpoC-dhfr plasmid obtained by inserting cDNA type EPO gene into vector pCMV-dhfr of claim 1 (KCTC 0180BP). 제2항 또는 제3항의 플라스미드로 안정하게 형질전환되어 인간 에리트로포이에틴을 생성하는 능력을 갖는포유류 유래 진핵세포인 형질전환체.A transformant which is a mammalian-derived eukaryotic cell having the ability to stably transform with the plasmid of claim 2 or 3 to produce human erythropoietin. 제4항에 있어서, pEpoC-dhfr 플라스미드로 형질전환된 것을 특징으로 하는 g2 형질전환체(KCTC 0183BP).The g2 transformant (KCTC 0183BP) according to claim 4, which is transformed with pEpoC-dhfr plasmid. 제4항에 있어서, pEpoC-dhfr 플라스미드로 형질전환되 것을 특징으로 하는 ECl 형질전환체(KCTC 0182BP).The ECl transformant (KCTC 0182BP) according to claim 4, characterized in that it is transformed with pEpoC-dhfr plasmid. 제4항에 있어서, 진핵세포가 dhfr CHO 변이 세포주인 것을 특징으로 하는 형질전환체.The transformant according to claim 4, wherein the eukaryotic cell is a dhfr CHO variant cell line. 제4항의 형질전환체를 배양하여 인간 에리트로포이에틴을 제조하는 방법.A method of producing human erythropoietin by culturing the transformant of claim 4. 제8항에 있어서, 배양액이 MTX가 함유된 α-MEM 배지인 것을 특징으로 하는 인간 에리트포포이에틴을 제조하는 방법.The method for producing human erythropoietin according to claim 8, wherein the culture solution is α-MEM medium containing MTX. 제9항에 있어서, MTX 농도를 20nM 이하로 유지하는 조건으로 세포주 g2~20을 배양하는 것을 특징으로 하는 인간 에리트로포이에틴을 제조하는 방법.The method for producing human erythropoietin according to claim 9, wherein the cell lines g2 to 20 are cultured under conditions of maintaining the MTX concentration at 20 nM or less. 제10항에 있어서, 세포주로 g2~20은 MTX 농도에서 적응된 g2 형질전환체인 것을 특징으로 하는 인간 에리트로포이에틴을 제조하는 방법.The method for preparing human erythropoietin according to claim 10, wherein the cell line g2-20 is a g2 transformant adapted at an MTX concentration. 제9항에 있어서, MTX 농도를 20nM 이하로 유지하는 조건으로 세포주 ECl~20을 배양하는 것을 특징으로 하는 인간 에리트로포이에틴을 제조하는 방법.The method for producing human erythropoietin according to claim 9, wherein the cell lines ECl to 20 are cultured under conditions of maintaining the MTX concentration at 20 nM or less. 제12항에 있어서, 세포주 ECl~20은 MTX 농도에서 적응된 ECl 형질전환체인 것을 특징으로 하는 인간 에리트로포이에틴을 제조하는 방법.13. The method of claim 12, wherein the cell lines ECl-20 are ECl transformants adapted at MTX concentration. ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.※ Note: The disclosure is based on the initial application.
KR1019950022441A 1995-07-27 1995-07-27 Preparation of erythropoietin KR0162021B1 (en)

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KR0162021B1 KR0162021B1 (en) 1998-11-16

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100431222B1 (en) * 2001-07-05 2004-05-12 (주) 하나환경 Sewage/waste water purging system form circulate
KR100493703B1 (en) * 2001-10-30 2005-06-02 신풍제약주식회사 Recombinant Vectors Useful in Animal Cells and Expression Vectors for Preparing Erythropoietin
WO2008048037A1 (en) * 2006-10-16 2008-04-24 Hanmi Pharmaceutical Co., Ltd. A novel vector and expression cell line for mass production of recombinant protein and a process of producing recombinant protein using same
WO2012057527A3 (en) * 2010-10-26 2012-07-26 Hanmi Science Co., Ltd. Method for mass production of factor vii/viia

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100620554B1 (en) 2004-06-05 2006-09-06 한국생명공학연구원 -72 Humanized Anti-TAG-72 Monoclonal Antibodies

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100431222B1 (en) * 2001-07-05 2004-05-12 (주) 하나환경 Sewage/waste water purging system form circulate
KR100493703B1 (en) * 2001-10-30 2005-06-02 신풍제약주식회사 Recombinant Vectors Useful in Animal Cells and Expression Vectors for Preparing Erythropoietin
WO2008048037A1 (en) * 2006-10-16 2008-04-24 Hanmi Pharmaceutical Co., Ltd. A novel vector and expression cell line for mass production of recombinant protein and a process of producing recombinant protein using same
KR100880509B1 (en) * 2006-10-16 2009-01-28 한미약품 주식회사 A Novel vector and expression cell line for mass production of recombinant protein and a process of producing recombinant protein using same
JP2010506586A (en) * 2006-10-16 2010-03-04 ハンミ ファーマシューティカル カンパニー リミテッド Novel vector and expression cell line for mass production of recombinant protein, and method for producing recombinant protein using the same
WO2012057527A3 (en) * 2010-10-26 2012-07-26 Hanmi Science Co., Ltd. Method for mass production of factor vii/viia

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