KR970059276A - Method for producing the vacuolar cytotoxin protein of Helicobacter pylori from recombinant E. coli - Google Patents

Method for producing the vacuolar cytotoxin protein of Helicobacter pylori from recombinant E. coli Download PDF

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KR970059276A
KR970059276A KR1019960001197A KR19960001197A KR970059276A KR 970059276 A KR970059276 A KR 970059276A KR 1019960001197 A KR1019960001197 A KR 1019960001197A KR 19960001197 A KR19960001197 A KR 19960001197A KR 970059276 A KR970059276 A KR 970059276A
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fusion protein
coli
protein
producing
gene
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KR1019960001197A
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KR100210508B1 (en
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장호진
유영준
김천형
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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
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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)
  • Health & Medical Sciences (AREA)
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Abstract

본 발명은 대장균을 이용하여 헬리코박터 필로리(Helicobacter pylori: H. pylori)의 항원 결정부위 단백직인 액포성 세포독소 단백질(Vacuolating Cytotoxin: Vac)을 융합단백질의 형태로 제조하는 방법에 관한 것으로, 헬리코박터 필로리 Vac의 168번째 아미노산부터 367번째 아미노산으로 구성된 단백질(V200)을 코딩하는 유전자에 유비퀴틴 유전자가 연결된 융합유전자를 포함하는 Ub-V200 융합단백질 발현벡터로 형질전환된 대장균을 융합단백질의 발현에 적합한 조건에서 배양하고, 융합단백질이 발현된 재조합 대장균 세포를 분쇄하고, 불용성 침전물을 트리톤이 함유된 완충용액으로 세척한 후, 우레아로 용해하고, 겔 여과 크로마토그래피 및 헤파린-세파로즈 크로마토그래피하는 단계를 포함하는 본 발명의 Ub-V200 융합단백질의 제조방법에 의하며 H. pylori 감염 진단과 백신 개발에 유용하게 사용될 수 있는 Ub-V200 융합단백질을 높은 수율로 대량 생산할 수 있다.The present invention relates to a method for producing a vacuolar cytotoxin (Vac), which is an antigenic determinant protein of Helicobacter pylori (H. pylori), in the form of a fusion protein using E. coli, Escherichia coli transformed with a Ub-V200 fusion protein expression vector containing a fusion gene in which a ubiquitin gene linked to a gene encoding a protein (V200) consisting of the 168th amino acid to the 367th amino acid of Laurie Vac is subjected to conditions suitable for the expression of the fusion protein , Pulverizing the recombinant E. coli cells expressing the fusion protein, washing the insoluble precipitate with a buffer solution containing triton, dissolving with urea, and performing gel filtration chromatography and heparin-sepharose chromatography Of H. pylori infection according to the method of producing the Ub-V200 fusion protein of the present invention The Ub-V200 fusion protein that can be useful for vaccine development can be mass-produced with high yield.

Description

재조합 대장균으로부터 헬리코박터 필로리의 액포성 세포독소 단백질 제조방법Method for producing the vacuolar cytotoxin protein of Helicobacter pylori from recombinant E. coli

본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음Since this is a trivial issue, I did not include the contents of the text.

제1도는 헬리코박터 필로리의 액포성 세포독소 단백질(Vac)의 단편 V200을 본 발명의 대장균 발현벡터 pETUB-V200으로 클로닝하는 과정을 도시한 것이고,FIG. 1 shows a process of cloning a fragment V200 of the vacuolar cytotoxin protein (Vac) of Helicobacter pylori with the E. coli expression vector pETUB-V200 of the present invention,

제2도는 본 발명의 융합단백질 유비퀴틴-V200의 발현을 변성 폴리아크릴아미드 겔 전기영동한 결과 (a) 및 유비퀴틴 단일 항체를 이용한 웨스턴 블롯팅한 결과 (b)로 확인한 결과를 나타낸 것이고,FIG. 2 shows the result of (a) the result of denaturing polyacrylamide gel electrophoresis of the fusion protein ubiquitin-V200 of the present invention and the result of Western blotting (b) using a ubiquitin single antibody,

제3도는 본 발명의 융합단백질 유비퀴틴-V200의 각 정제단계에서, 변성 폴리아크릴아미드 겔 전기영동한 결과 (a) 및 전기영동 후 헬리코박터 필로리 감염자의 혈청을 이용하여 웨스턴 블롯팅한 결과 (b)를 나타낸 것이다.FIG. 3 shows the results (a) of denaturing polyacrylamide gel electrophoresis and Western blotting (b) using serum of a patient infected with Helicobacter pylori after electrophoresis in each purification step of the fusion protein ubiquitin-V200 of the present invention. .

Claims (6)

헬리코박터 필로리(Helicobacter pylori: H. pylori) 액포성 세포독소 단백질(Vacuolating Cytotoxin: Vac)의 168번째 아미노산부터 367번째 아미노산으로 구성된 단백질(V200)을 코딩하는 유전자에 유비퀴틴 유전자가 연결된 융합유전자.A fusion gene in which a ubiquitin gene is linked to a gene encoding a protein (V200) consisting of the 168th amino acid to the 367th amino acid of Helicobacter pylori (H. pylori) vacuolar cytotoxin (Vac). 제1항의 융합유전자를 포함하는 유비퀴틴-액포성 세포독소 단백질(Ub-V200) 융합단백질 발현벡터.(Ub-V200) fusion protein comprising the fusion gene of claim 1. 제2항에 있어서, 발현벡터 pETUB-V200.3. The method according to claim 2, wherein the expression vector pETUB-V200. 제2항의 발현벡터로 형질전환된 대장균.An E. coli transformed with the expression vector of claim 2. 제4항에 있어서, 대장균 BL21/(DE3)/pLys,S/pETUB-V200.The method according to claim 4, wherein the Escherichia coli BL21 / (DE3) / pLys, S / pETUB-V200. 제4항의 대장균을 융합단백질의 발현에 적합한 조건에서 배양하고, 융합단백질이 발현된 재조합 대장균 세포를 분쇄하고, 불용성 참전물을 트리톤이 함유된 완충용액으로 세척한 후, 우레아로 용해하고, 겔 여과 크로마토그래피 및 헤파린-세파로즈 크로마토그래피하는 단계를 포함하는 Ub-V200 융합단백질의 제조방법.Culturing the Escherichia coli of claim 4 under conditions suitable for the expression of the fusion protein, pulverizing the recombinant E. coli cells expressing the fusion protein, washing the insoluble cells with a buffer solution containing triton, dissolving with urea, ≪ / RTI > chromatography and heparin-sepharose chromatography. ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.※ Note: It is disclosed by the contents of the first application.
KR1019960001197A 1996-01-20 1996-01-20 Process for the preparation of vacuolating cytotoxin of helicobacter pyroli in e.coli KR100210508B1 (en)

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Cited By (1)

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Publication number Priority date Publication date Assignee Title
KR100845043B1 (en) * 2006-07-25 2008-07-08 경북대학교 산학협력단 A pool of recombinant Helicobacter pylori vacuolating cytotoxin fragments from E.coli for vaccine

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IT202100015653A1 (en) 2021-06-15 2022-12-15 Ga Vo Mecc S R L METHOD AND APPARATUS FOR REMOVING SHEET MATERIAL FROM A TUBULAR CORE ON WHICH SAID SHEET MATERIAL IS ROLLED

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100845043B1 (en) * 2006-07-25 2008-07-08 경북대학교 산학협력단 A pool of recombinant Helicobacter pylori vacuolating cytotoxin fragments from E.coli for vaccine

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