金黄色葡萄球菌α-溶血素基因表达及溶血活性检测

The expression of the gene encoding α-hemolysin of Staphylococcus aureus and termination of its hemolytic activity

目的为了进一步研究金黄色葡萄球菌(Staphylococcus aureus)α-溶血素(Hla)的分子致病机理及其抗原性,对S.aureusHla进行了表达纯化。方法用聚合酶链式反应(PCR)技术,从S.aureuswood46株中扩增出hla基因,将该基因经BamHI和SalI双酶切后克隆到表达载体PET-32a中,获得重组质粒pET-32a(+)/hla,并在大肠杆菌BL21(DE3)中诱导表达,对表达产物进行SDS-PAGE电泳和Western blot检测分析,然后进行纯化和用溶血试验检测其活性。结果扩增的hla基因序列与已发表的hla基因序列同源性为100%,构建的重组表达质粒pET-32a(+)/hla在E.coliBL21(DE3)中得到表达,重组α-溶血素蛋白为53kDa,纯化后的溶血活性为5000HU。结论成功表达并获得了有活性的重组Hla蛋白。

In order to characterize the antigenicity in more detail and the molecular pathogenesis of Staphylococcus aureus α-hemolysin（Hla）, the gene encoding hla from S. aureus Wood46 strain was amplified with PCR and cloned to expression vector pET-32a after double digestion with BamHI and SalI to obtain the recombinant plasmid pET32a（＋）/hla. After sequencing, this recombinant plasmid was then inserted into E. coli BL21（DE3） to express the recombinant protein. The expressed recombinant protein Hla was analyzed with SDS-PAGE and Western blotting, purified with Ni-particles. And its hemolytic activity was determined. It was found that the nucleotide sequence homology of the amplified hla gene with other published hla genes was 100%, and the molecular mass of the expressed recombinant protein was 53 kDa. The hemolytic activity of the puri-fied protein appeared to have 5000 hemolytic unit （HU）.