【摘要】 目的克隆表达粪肠球菌溶血素cylL基因,为制备单抗、开发疫苗及其致病机制研究奠定基础。方法从粪肠球菌中扩增溶血素cylL基因,相应酶切后,克隆到原核表达载体pET42a中,构建pET-cylL重组质粒。将pET-cylL质粒转化入大肠杆菌BL21(DE3)。经KpnI、XhoI酶切及测序鉴定,以IPTG诱导表达融合蛋白,用SDS-PAGE、Western blot进行分析。结果PCR体外扩增cylL基因产物约206bp,成功构建了重组表达质粒pET-cylL;SDS-PAGE、Western免疫印迹显示蛋白表达带的分子量约为39.6kD。结论成功构建粪肠球菌溶血素cylL基因的重组表达质粒,并在大肠杆菌BL21(DE3)中表达。更多还原

【Abstract】 To clone and express Enterococus faecalis cylL gene in order to provide the basis for preparation of monoclonal antibody and development of enterococcus vaccine as well as further studing the pathogenetic mechanisms, cylL gene was amplified from Enterococus faecalis by PCR. After digestion with restriction enzymes, the PCR products were cloned into prokaryotic expression vector pET42a and then transformed to E.coli BL21(DE3).The recombinant plasmid pET-cylL was identified by KpnI,XhoI restriction endonuclease digestion and DNA sequencing. Expression of the fusion protein was induced by IPTG, and analyzed by SDS-PAGE and Western blotting. Results showed that cylL gene of 206 bp was amplified from Enterococus faecalis and the recombinant plasimid pET-cylL was successfully constructed.The expressed product was found to be 39.6 kDa in molecular weight as demonstrated by SDS-PAGE and Western blotting. It was concluded that Enterococus faecalis hemolysin cylL recombinant plasimid was successfully constructed and expressed in E.coli BL21(DE3).更多还原