金黄色葡萄球菌甲氧西林耐药辅助基因femB的克隆和原核表达

Cloning and Prokaryotic Expression of femB Gene of Staphylococcus aureus

金黄色葡萄球菌femB基因与甲氧西林高水平耐药密切相关,可能成为开发抗MRSA药物的新靶位.以金葡菌基因组DNA为模板,PCR扩增femB全长基因,所得片段与pGM-T载体连接并转化感受态大肠杆菌DH5α,阳性克隆以PCR、双酶切及测序鉴定.将鉴定正确的目的片段定向克隆到pGEX-4T-1表达载体中,转化至大肠杆菌BL21后经IPTG诱导表达GST/FemB融合蛋白;采用SDS-PAGE及Western blot对融合蛋白进行鉴定.结果显示,重组质粒在宿主菌中获得了高效表达,融合蛋白相对分子质量为75 kD,该融合蛋白可与抗GST-tag抗体特异结合;表明femB基因的原核表达系统构建成功,为进一步研究其生物学功能奠定了基础.

FemB gene is closely related with high level of methicillin resistance of Staphylococcus aureus（S. aureus） and is a potential new target for antibiotics against MRSA. Genomic DNA from S. aureus was used as template for PCR amplification offemB gene. The obtained femB gene fragment was cloned into pGM-T vector and then transformed into the competent E. coli DHSa. The positively clone was identified by PCR, restriction endonuclease analysis and DNA sequencing. The identified fragment was cloned into expression vector pGEX-4T-1 and transformed into E. coli BL21, and then induced by IPTG to express GST/FemB fu- sion protein. The expressed protein was identified by SDS-PAGE and Western blot. Results showed that femB gene was cloned and recombinant plasmid was constructed successfully. The recombinant protein with a relative molecular mass 75 kD, which specifically combined with the anti-GST-tag antibody, was highly expressed in E.coli. A prokaryotic expression system of f craB gene has been constructed successfully, which laid foundations for investigation on its biological functions.