金黄色葡萄球菌α溶血素的原核表达及其生物活性分析

摘要：α溶血素（α-Hemolysin, α-HL）是金黄色葡萄球菌的主要毒力因子之一，利用PCR技术从金黄色葡萄球菌临床分离株扩增出编码α溶血素的基因(α-HL)，经克隆筛选和测序鉴定后，构建成该基因的原核表达质粒pET32a+-α-HL。经诱导表达后，进行SDS-PAGE分析，结果表明:α-HL在大肠杆菌中已融合表达,融合蛋白的分子量为53kD。溶血实验证明该融合蛋白具有较强的溶血作用,其溶血效价达2.26×104 HU/mg,表明表达产物具有生物活性,这为进一步研究其致病与免疫机理、疫苗设计提供了条件。

Prokaryotic Expression and Bioactivity of the Staphylococcal α-hemolysin

Abstract: To express the Staphylococcal α-Hemolysin gene in E. coli and study its primary biological characterristics, α-Hemolysin (α-HL) gene without signal peptide sequence was amplified from Staphylococcus aureus by PCR and inserted into pMD18-T vector. The cloned α-HL gene was then inserted into prokaryotic expression vector pET32a+ and transformed into E. coli BL21. The predicted protein was detected by SDS-PAGE after IPTG induction, which had molecular weight approximately 53 kD. Hemolytic experiment demonstrated the expressed protein can lyse mouse red cell and its hemolytic titer attained 2.26 ×104 HU/mg. Conclusively, the Staphylococcal α-HL gene was successfully cloned and expressed in E. coli. The successful expression of α-HL in E. coli BL21 constituted a solid foundation for further researches such as pathogenesis and immune mechanism and vaccine development.