金黄色葡萄球菌β-溶血素的原核表达及其溶血活性检测  

Prokaryotic expression of Staphylococcus aureus β-hemolysin and the hemolytyic activity of the expressed recombinant protein

作　　者：李冬野[1] 崔玉东[1] 侯喜林[1] 朱战波[1] 朴范泽[1] 

机构地区：[1]黑龙江八一农垦大学,大庆163319

出　　处：《中国人兽共患病学报》2007年第2期136-139,共4页Chinese Journal of Zoonoses

基　　金：黑龙江省科技攻关重大项目(GA02B501)

摘　　要：目的为进一步研究金黄色葡萄球菌β-溶血素的致病性及免疫保护作用,对金黄色葡萄球菌β-溶血素进行了表达。方法应用聚合酶链式反应(PCR)技术,从金黄色葡萄球菌中扩增出β-溶血素基因,将该基因克隆到原核表达载体PET-32a中,获得重组质粒PET-32aβ-,并在大肠杆菌BL21(DE3)中进行诱导表达。对表达产物进行SDS-PAGE电泳和Westernblot检测分析,并对纯化后表达产物进行平板溶血试验,检测其溶血活性。结果PCR扩增的β-溶血素基因片段全长为982bp;表达的β-溶血素重组蛋白大小为57kD;纯化的重组蛋白可使绵羊红细胞发生明显溶血。结论结果表明所克隆的β-溶血素基因在大肠杆菌中得到了良好的表达,并保持良好的溶血活性。In order to study the pathogenesis of Staphulococcus aureus infection in more detail and to investigate the immunogenicity of the S. aureus-hemolysin, the gene encoding the β-heemolysin （h/b） was amplified by PCR from a strain of S. aureus and cloned to the vector pET-32 to obtain the recombinant plasmid pET-32-β which was expressed in E. coli BL21（DE3） cells with induction. Then the expressed products were analyzed with SDS-PAGE and Western blotting, and subjected to the analysis with hemolytic testing on plates after purification to detect its hemolytic activity. It was shown that the full length of the amplified gene fragment of β-hemolysin was 982 bps. The fusion protein of β-hemolydin was highly expressed in E. coli BL21 （DE3） cells and its molecular weight was about 57 kDa. In addition, this protein could induce obvious hemolysis of sheep erythrocytes as demonstrated by the hemolytic testing on plates. These results indicate that the cloned gene of β-hemolysin is well expressed in E. coli and the expressed β-hemolysin shows good hemolytic activity.

关 键 词：金黄色葡萄球菌 β-溶血素(hlb)基因 原核表达 WESTERN BLOT 

分 类 号：R378.1[医药卫生—病原生物学]