Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Construction and Characterization of the Vibrio parahaemolyticus Collagenase Inactivated Mutant

Vibrio parahaemolyticus collagenase 불활성화 돌연변이체의 제조 및 특성

  • 이재원 (부산대학교 자연과학대학 미생물학과) ;
  • 전인준 (부산대학교 자연과학대학 미생물학과) ;
  • 강호영 (부산대학교 자연과학대학 미생물학과) ;
  • 차재호 (부산대학교 자연과학대학 미생물학과)
  • Published : 2004.04.01
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Abstract

For better understanding of the host infection mechanism of Vibrio, a Vibrio parahaemolyticus collagenase mutant was generated by insertional inactivation of a vppC gene encoding extracellular collagenase. A recombinant DNA containing vppC::nptII was cloned into a suicide plasmid pDMS197, resulted in pVCM03. The recombinant suicide plasmid pVCM03 contained in E. coli $\chi$7213 was transferred to a wild-type V. parahaemolyticus 04 through conjugation. The recombinant vppC::nptII DNA in pVCM03 was exchanged with wild-type allele by homologous recombination resulting vppC mutant, V. parahaemolyticus CM. The mutant was selected and screened on TCBS media containing 10% sucrose and kanamycin. The mutation by allele exchange was confirmed with the comparison of the size of DNAs amplified by PCR. V. parahaemolyticus CM showed at least 4-fold less collagen-degrading activity than those of wild-type, and the mutant exhibited less cytotoxicity than that of wild-type in MTT assay.

장염비브리오균의 숙주 내 감염을 일으키는 기작을 이해하기 위하여 세포외 효소 중의 하나인 콜라겐분해효소의 유전자가 불활성화된 돌연변이체를 제작하였다. 콜라겐분해효소의 유전자인 vppC 유전자에 항생제 내성 유전자인 nptII를 삽입하여 제작된 재조합 DNA를 suicide vector인 pDMS197에 클로닝하여 pVCM03이라 명명하였다. 재조합 suicide 플라스미드 pVCM03을 E. coli 7213에 형질전환하여 접합을 통하여 원 균주인 V. varahaemolyticus 04에 전달하였다. 전달된 pVCM03 유래의 재조합 vvpC::npfII DNA는 homologous recombination에 의해 wild-type allele와 교환되어 돌연변이체를 형성하게 되고, 돌연변이체는 10% sucrose가 함유된 TCBS 배지에서 선별되었다. Allele exchange는 PCR에 의한 증폭된 DNA의 크기 비교로 확인하였다. 돌연변이체인 V. parahaemolyticus CM은 원 균주와 비교하였을 때 약 4배정도 낮은 콜라겐 분해 활성을 나타내었고, vero cell을 이용한 MTT assay에서도 원 균주에 비하여 낮은 세포독성을 보였다.

Keywords

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