超抗原SEA的基因克隆、原核表达与鉴定

目的：构建pRSET-SEA重组表达载体，转化大肠杆菌BL21(DE3)pLysS，诱导表达超抗原葡萄球菌肠毒素A(staphylococcal enterotoxin A，SEA)，进行分离、纯化及western bolt鉴定。方法：采用PCR技术，从产SEA的葡萄球菌标准菌株FRI100基因组DNA中获得SEA全长序列，克隆人pUC19中，进行测序，构建pRSET-SEA表达质粒，转化大肠杆菌BL21(DE3)pLysS，通过异丙基硫代-β-D-半乳糖苷（isopropyl-beta-D-thiogalactopyranoside，IPTG）诱导表达，分离、纯化及western blot鉴定。结果：PCR获得超抗原SEA基因片段，DNA测序结果与文献报道一致；构建了pRSET-SEA表达质粒，并成功地诱导表达出32000u的蛋白；Western blot鉴定所得蛋白能够与SEA单克隆抗体特异性结合。结论：本研究成功地克隆了SEA全长，并进行了原核表达和分离、纯化，获得了SEA蛋白。

Cloning, prokaryotic expression and identification of superantigen SEA gene

Objective To clone the SEA gene and construct the prokaryotic expression vector pRSET SEA. The induced protein was purified and identified by western blot.Methods To clone the SEA gene from standard Staphylococcus aureus FRI 100, and construct the prokaryotic expression plasmid pRSET SEA. E. coli BL21(DE3)pLysS transformed with plasmid pRSET SEA was induced by IPTG. The induced protein was purified by Ni 2+ NTA system and identified by western blot.Results We cloned the SEA gene and its sequence was the same as reported in literature. E. coli BL21(DE3)pLysS contained pRSET SEA can express a 32 000 u protein which can specially bind with the anti SEA mAb. Conclusion In this study, the SEA gene was cloned and expressed in E. coli successfully, and the induced protein was purified and identified.