Journal of Veterinary Clinics (한국임상수의학회지)

The Korean Society of Veterinary Clinics (한국임상수의학회)
권호 표지

Induction of Deletion Mutation for the Enzymatic Domain in the Shigatoxin2e A Subunit Gene of Esherichila coli O139 Isolates and Expression of Mutated Protein

분리 대장균 O139의 Shigatoxin2e A 유전자의 효소 활성부에 대한 결손변이 유발 및 변이 단백질의 발현

  • Cho Eun-jung (Gyongnam Livestock Promotion Institute) ;
  • Kim Do-kyong (Gyongnam Livestock Promotion Institute) ;
  • Kim Sang-hyun (Department of Laboratory Medicine and Pathology, Biochemistery, University of Toronto) ;
  • Kim Yeong-il (National Veterinary Research and Quarantine Service) ;
  • Lee Chul-hyun (National Veterinary Research and Quarantine Service) ;
  • Lee Woo-won (Busan Institute of Health and Environment) ;
  • Son Won-geun (Department of Veterinary Medicine, Jeju National University) ;
  • Shin Jong-Uk (College of Veterinary Medicine, Research Institute of Life Science, Gyeongsang National University) ;
  • Kim Yong-hwan (College of Veterinary Medicine, Research Institute of Life Science, Gyeongsang National University)
  • 조은정 (경상남도 축산진흥연구소) ;
  • 김도경 (경상남도 축산진흥연구소) ;
  • 김상현 (캐나다 구엘프 대학교 수의과대학) ;
  • 김영일 (수의과학 검역원) ;
  • 이철현 (수의과학 검역원) ;
  • 이우원 (부산시 보건환경연구원) ;
  • 손원근 (제주대학교 농과대학 수의학과) ;
  • 신종욱 (경상대학교 수의과대학 생명과학연구소) ;
  • 김용환 (경상대학교 수의과대학 생명과학연구소)
  • Published : 2005.12.01
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Abstract

This study was done to produce a mutated protein inactivated cytotoxicity of Shigatoxin 2e (Stx2e) of E.coli O139 isolates by deletional mutagenesis of Stx2e A subunit gene encoding active-site cleft of enzymatic domain in ST2e holotoxin. Cytotoxicity of the toxoid expressed from the mutant Stx2e gene was compared with wild type Stx2e for development of vaccine candidate. A recombinant plasmid pED18 containing Stx2e gene ot E.coli O139 isolates was used to generate mutation plasmid. Deletion mutagenesis was conducted for Stx2e A subunit gene encoding enzymatically active domain by polymerase chain reaction (PCR) using ot designed primer to induce deletional mutation. DNA sequence analysis was confirmed that the pentamer (Typ 202- Ser 206) that lies within the proposed active-site cleft in the second region was completely deleted. A DNA fragment of 1.1 kb that encode the new mutant Stx2eA gene was inserted into plasmid pRSET vector digested with EcoRV-Hind III and named pEDSET The PEDSET was transformed in E. coli for expression of mutant protein and the protein was confirmed by SDS-PACE and Western-blotting. The protein expressed by the mutant was tested to confirm the reduction of cytotoxic activities on Vero cell using microcytotoxicity assay compared with wild type Stx2e, the cytotoxicity of deletional mutant protein was at least reduced by 3,000-fold on Vero cell.

Keywords

References

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