超抗原SEA的基因克隆、原核表达与鉴定  

Cloning, prokaryotic expression and identification of superantigen SEA gene

作　　者：叶菁[1] 隋延仿[1] 李增山[1] 陈广生[1] 张秀敏[1] 曹云新[2] 

机构地区：[1]第四军医大学基础部病理学教研室,陕西西安710032 [2]第四军医大学基础部免疫学教研室,陕西西安710032

出　　处：《免疫学杂志》2002年第6期418-420,共3页Immunological Journal

基　　金：国家自然科学基金 (39770 82 7);全军医药卫生科研基金 (0 1Z0 84)重点资助项目

摘　　要：目的　构建pRSET SEA重组表达载体 ,转化大肠杆菌BL2 1 (DE3)pLysS ,诱导表达超抗原葡萄球菌肠毒素A(staphylococcalenterotoxinA ,SEA) ,进行分离、纯化及westernblot鉴定。方法　采用PCR技术 ,从产SEA的葡萄球菌标准菌株FRI1 0 0基因组DNA中获得SEA全长序列 ,克隆入pUC1 9中 ,进行测序 ,构建pRSET SEA表达质粒 ,转化大肠杆菌BL2 1 (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westernblot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32 0 0 0u的蛋白 ;Westernblot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。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.

关 键 词：葡萄球菌肠毒素A 超抗原 原核表达 PCR 鉴定 

分 类 号：R392.11[医药卫生—免疫学]