KR980002259A - Method for producing species-specific protein antigen of Helicobacter pylori from recombinant E. coli - Google Patents

Method for producing species-specific protein antigen of Helicobacter pylori from recombinant E. coli Download PDF

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KR980002259A
KR980002259A KR1019960020906A KR19960020906A KR980002259A KR 980002259 A KR980002259 A KR 980002259A KR 1019960020906 A KR1019960020906 A KR 1019960020906A KR 19960020906 A KR19960020906 A KR 19960020906A KR 980002259 A KR980002259 A KR 980002259A
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ssa
fusion protein
coli
protein
histidine
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KR1019960020906A
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KR0177304B1 (en
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장호진
김천형
황상연
유영준
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성재갑
주식회사 Lg 화학
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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
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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
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    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)

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  • Chemical & Material Sciences (AREA)
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  • Gastroenterology & Hepatology (AREA)
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Abstract

본 발명은 대장균을 이용하여 헬리코박터 필로리(Helicobacter pylori : H. pylori)의 항원 결정부위 단백질인 종특이 단백질 항원(Species-Specific Protein Antigen : SSA)을 융합단백질의 형태로 제조하는 방법에 관한 것으로, 헬리코박터 필로리의 SSA 유전자에 히스티딘과 유비퀴틴 유전자가 연결된 융합유전자를 포함하는 His-Ub-SSA 융합단백질 발현벡터로 형질전환된 대장균을 융합단백질의 발현에 적합한 조건에서 배양하고, 융합단백질이 발현된 재조합 대장균 세포를 구아니딘 염이 포함된 완충용액에서 분쇄하고, 원심분리한 상등액을 히스티딘 결합수지 친화성 크로마토그래피하는 단계를 포함하는 본 발명의 His-Ub-SSA 융합단백질의 제조방법에 의하면 H. pylori 감염 진단과 백신 개발에 유용하게 사용될 수 있는 His-Ub-SSA 융합단백질을 높은 수율로 대량 생산할 수 있다.The present invention relates to a method for preparing a species-specific protein antigen (SSA), an antigenic determinant protein of Helicobacter pylori (H. pylori) using E. coli, in the form of a fusion protein. E. coli transformed with a His-Ub-SSA fusion protein expression vector comprising a fusion gene linked to histidine and ubiquitin genes to the SSA gene of Helicobacter pylori was cultured under conditions suitable for expression of the fusion protein, and the recombinant protein expressed with the fusion protein was expressed. According to the method for preparing the His-Ub-SSA fusion protein of the present invention, the method comprises the steps of pulverizing Escherichia coli cells in a buffer containing guanidine salt and subjecting the supernatant centrifuged to histidine-binding resin affinity chromatography. High-yield production of His-Ub-SSA fusion protein, which can be useful for diagnosis and vaccine development. All.

Description

재조합 대장균으로부터 헬리코박터 필로리의 종특이 단백질 항원 제조방법Method for producing species-specific protein antigen of Helicobacter pylori from recombinant E. coli

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

제1도는 종특이 단백질 항원(SSA) 유전자를 본 발명의 대장균 발현벡터 pETHUB-SSA로 클로닝하는 과정을 도시한 것이다.Figure 1 shows the procedure for cloning a species-specific protein antigen (SSA) gene into the E. coli expression vector pETHUB-SSA of the present invention.

제2도는 본 발명의 발현벡터 pETHUB-SSA로 형질전환된 대장균을 배양하여 얻은 세포 침전물을 변성 폴리아크릴아미드 겔 전기영동한 후 유비퀴틴 단일 항체를 이용하여 웨스턴 블롯팅(western blotting)한 결과를 나타낸 것이다.Figure 2 shows the result of Western blotting using a ubiquitin single antibody after denatured polyacrylamide gel electrophoresis of the cell precipitate obtained by culturing E. coli transformed with the expression vector pETHUB-SSA of the present invention .

제3도는 본 발명의 발현벡터 pETHUB-SSA로 형질전환된 대장균을 배양하여 얻은 세포 침전물을 변성 폴리아크릴아미드 겔 전기영동한 결과로서, IPTG로 발현 유도하기 전후를 비교한 것이다.3 is a result of denaturing polyacrylamide gel electrophoresis of cell precipitates obtained by culturing E. coli transformed with the expression vector pETHUB-SSA of the present invention.

Claims (6)

헬리코박터 필로리(Helicobacter pylori:H. pyori)의 종특이 단백질 항원(Species-Specific Protein Antigen:SSA) 유전자에 히스티딘과 유비퀴틴 유전자가 연결된 융합유전자.A fusion gene in which histidine and ubiquitin genes are linked to the Species-Specific Protein Antigen (SSA) gene of Helicobacter pylori (H. pyori). 제1항의 융합유전자를 포함하는 히스티딘-유비퀴틴-종특이 단백질 항원 (His-Ub-SSA) 융합단백질 발현벡터.A histidine-ubiquitin-species-specific protein antigen (His-Ub-SSA) fusion protein expression vector comprising the fusion gene of claim 1. 제2항에 있어서, 발현벡터 pETHUB-SSA.The expression vector pETHUB-SSA of claim 2. 제2항의 발현벡터로 형질전환된 대장균.E. coli transformed with the expression vector of claim 2. 제4항에 있어서, 대장균 BL21(DE3) pLYS.S(pET HUB SSA).The E. coli BL21 (DE3) pLYS.S (pET HUB SSA) according to claim 4. 제4항의 대장균을 융합단백질의 발현에 적합한 조건에서 배양하고, 융합단백질이 발현된 재조합 대장균 세포를 구아니딘 염이 포함된 완충용액에서 분쇄하고, 원심분리한 상등액을 히스티딘 결합수지 친화성 크로마토그래피하는 단계를 포함하는 His-Ub-SSA 융합단백질의 제조방법.E. coli is cultured under conditions suitable for the expression of the fusion protein, the recombinant E. coli cells expressing the fusion protein is pulverized in a buffer solution containing guanidine salt, and the centrifuged supernatant is subjected to histidine binding resin affinity chromatography Method for producing a His-Ub-SSA fusion protein comprising a. ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.※ Note: The disclosure is based on the initial application.
KR1019960020906A 1996-06-12 1996-06-12 Method for producing species specific protein antigen of Helicobacter pylori from recombinant E. coli KR0177304B1 (en)

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