KR940021731A - Crm 단백질 및 디프테리아 독소의 생산을 위한 방법 및 플라스미드 - Google Patents

Crm 단백질 및 디프테리아 독소의 생산을 위한 방법 및 플라스미드 Download PDF

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KR940021731A
KR940021731A KR1019940004356A KR19940004356A KR940021731A KR 940021731 A KR940021731 A KR 940021731A KR 1019940004356 A KR1019940004356 A KR 1019940004356A KR 19940004356 A KR19940004356 A KR 19940004356A KR 940021731 A KR940021731 A KR 940021731A
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crm
plasmid
diphtheria toxin
protein
corynebacterium
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제이 메트 카프 벤자민
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알퐁스 아아르노
아메리칸 사이아나밋드 캄파니
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    • C12N15/77—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Corynebacterium; for Brevibacterium
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    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)

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Abstract

본 발명은 CRM 197 단백질 디프테리아 독소 및 디프테리아 독소와 관련된 기타 CRM 단백질들의 풍부한 양을 생산하기 위한 새로운 방법 및 플라스미드 시스템, 뿐만아니라 상기 새로운 플라스미드에 의해 형질전환된 미생물에 관한 것이다. 특히 바람직한 DNA 플라스미드, 즉 플라스미드 pNG2-22 및 비독소원베타파지로부터의 CRM197에 대한 유전자를 조합한 명칭 pPX 3511을 서술한다. 새로운 플라스미드 시스템은 코리네박테리움 디프테리아(Corynebacterium diphtheriae)의 균주를 다수 리조겐의 사용없이 높은 수준의 CRM 197 단백질을 발현할 수 있는 균주로 형질전환시킬 수 있다. 본 발명은 예컨대 프로모터 강도를 증가시키거나 철 조절로 프로모터를 제거하므로써 발현벡터를 조작할 필요없이 단백질 생산을 증가시키기 위한 정밀한 수단을 제공한다.

Description

CRM 단백질 및 디프테리아 독소의 생산을 위한 방법 및 플라스미드
본 내용은 요부공개 건이므로 전문내용을 수록하지 않았음
제1도는 재조합 DNA 플라스미드, 명칭 pPX3511이다,
제2도는 시이. 디프테리아(C.diphtheriae) C7의 상이한 균주로부터 CRM197(61.8 킬로달톤)의 생산을 나타내는 12% SDS-PAGE 겔이다,
제3도는 항생제 선택성이 없이 플라스미드 보유의 지시물로서 클로람페니콜 내성을 사용하여 시이. 디프테리아 C7(β197)tox-내 플라스미드 pPX3511의 안정성을 나타낸다.

Claims (10)

  1. 하기로 구성되는, 디프테리아 독소와 교차 반응성인 CRM 단백질 또는 디프테리아 독소의 생산 방법 : a) 디프테리아 독소 또는 CRM 단백질을 코딩하는 유전자; b) 복제의 코리네박테리움(Corynebacterium)원천; 및 c) 선택가능한 마커를 함유하는 프라스미드로써 코리네박테리움 종들의 미생물을 형질전환시키고, 미생물이 유전자를 발현하기에 충분한 조건하에서 상기 독소 또는 단백질을 발현시킨다.
  2. 제1항에 있어서, 형질전환 방법이 일렉트로포레이션에 의한 방법.
  3. 제1항에 있어서, 미생물이 코리네박테리움 디프테리아(Corynebacterium diphtheriae)인 방법.
  4. 제3항에 있어서, 코리네박테리움 디프테리아의 균주가 C7 균주인 방법.
  5. 제1항에 있어서, CRM 유전자는 CRM 197, CRM 45, CRM 30, CRM 228 및 CRM 176으로 구성된 군으로부터 선택된 방법.
  6. 제1항에 있어서, 복제의 원천이 코리네박테리움 플라스미드 pNG2로부터 유래된 방법.
  7. 하기 a)-c)로 구성되는, 숙주내에서 디프테리아 독소와 교차 반응성인 CRM 단백질 또는 디프테리아 독소를 발현하기 위한 플라스미드 : a) 디프테리아 독소 또는 CRM 단백질을 코딩하는 유전자; b) 복제의 코리네박테리움 원천; 및 c) 임의로 다수 클로닝 부위에 연결된 선택가능한 마커.
  8. 제7항에 있어서, 단백질을 코딩하는 유전자가 하나이상의 단백질, 펩티드 또는 그의 에피토프를 코딩하는 뉴클레오티드 서열에 작동가능하게 연결된 플라스미드.
  9. ATCC 수탁번호 75415인 플라스미드 pPX 3511.
  10. 제7항의 플라스미드로써 형질전환된 코리네박테리움 종들의 미생물.
    ※ 참고사항 : 최초출원 내용에 의하여 공개하는 것임.
KR1019940004356A 1993-03-05 1994-03-04 Crm단백질및디프테리아독소의생산을위한방법및플라스미드 KR100316004B1 (ko)

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