【摘要】 研究了黄杆菌 ATCC2 75 5 1的 opd基因在大肠杆菌中的表达 ,并对于表达产物进行了胞内定位和酶活测定 .通过 PCR扩增和 PCR产物直接克隆 ,将黄杆菌质粒上的一段长达 1137bp的 opd基因进行扩增及克隆 ,然后按正确的读码框亚克隆于表达载体 p ET2 8b(+ )上 ,转化宿主菌 E.coli BL 2 1(DE3) ,经 IPTG诱导 ,获得了较高量的基因表达产物 .该表达产物以包涵体的形式存在于细胞内 ,通过分离包涵体可以得到电泳较纯的条带 .工程菌的细胞活性达到原始菌株黄杆菌的水平更多还原

【Abstract】 The cloning and expression of a Flavobacterium -derived opd gene in E.coli was studied. The 1137 bp DNA fragment encoding organophosphorus hydrolase was amplified with PCR, then directly cloned into pGEM-T vector. Plasmid pET-opd was constructed by subcloning the opd gene into plasmid pET28b(+) in correct reading frame. The expression product could be observed on an SDS-PAGE gel as a protein band with molecular weight of about 38×10 3. In the cell lysate induced with IPTG, the target protein located intracellular and aggregated into the inclusion bodies. Compared with the native Flavobacterium , the recombinant E.coli cells showed a similiar activity of organophosphorus hydrolase.更多还原