분리 대장균 O139의 Shigatoxin2e A 유전자의 효소 활성부에 대한 결손변이 유발 및 변이 단밸질의 발현 = Induction of deletion mutation for the enzymatic domain in the shigatoxin2e A subunit gene of Esherichila coli o139 isolates and expression of mutated protein

다국어 초록 (Multilingual 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 of 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 of 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-HindIII and named pEDSET. The pEDSET was transformed in E.coli for expression of mutant protein and the protein was confirmed by SDS-PAGE 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.

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