金黄色葡萄球菌β-溶血素基因的克隆及原核表达载体的构建

Cloning and construction of prokaryotic expression vector for the beta-hemolysin of Staphylococcus aureus

根据GenBank中的金黄色葡萄球菌β-溶血素(hlb)基因序列,设计合成1对引物,采用PCR方法扩增出与预期设计大小相符的hlb基因特异性条带,将扩增出的DNA片段克隆到pMD18-T载体中,转化感受态细胞,经平板筛选,酶切鉴定,获得阳性重组质粒,对重组质粒进行序列测定,结果表明:金黄色葡萄球菌β-溶血素基因全长982bp,不含终止密码子的目的基因,插入的位点、大小与读码框均正确。将hlb基因插入到原核表达pET-32a中,转化到BL-21感受态细胞中,获得了表达hlb基因的阳性重组质粒。

One pair of specific primers to hlb gene of S. aureus was designed and synthesized according to the published sequence in GenBank, a DNA encoding beta-hemolysin（hlb）was amplified by PCR and inserted it into pMDlg-T. The recombinant plasmid o（ pMD-T-β was transformed into host BL-21 bacterium. The recombinant plasmid of pET-32a-βcontaining hlb gene were identified by restriction enzymtic analysis, PCR examination and sequence analysis. The result indicates that the recombinant PET-32a-β prokaryotic expression vector has been successfully construted.