金黄色葡萄球菌肠毒素E的克隆表达及其生物活性研究

 目的克隆金黄色葡萄球菌肠毒素E(SEE)基因,获得重组SEE蛋白(rSEE),并对rSEE进行生物活性分析。方法通过PCR从金黄色葡萄球菌FR1326菌株基因中得到肠毒素SEE的成熟肽基因,将其克隆至融合表达载体pGEX-4T-1,转化大肠杆菌进行表达并对融合蛋白进行亲和色谱纯化,通过MTF法研究其生物活性。结果成功构建了SEE的表达载体,并得到高纯度的重组蛋白。MTY法结果表明,rSEE具有良好的促淋巴细胞增殖的能力以及肿瘤细胞的杀伤活性。结论具超抗原活性和高纯度的rSEE,为进一步研究该蛋白的抗肿瘤机制,构建靶向抗肿瘤融合蛋白奠定了基础。

Study on Expression and Biological Activity of Staphylococcal Enterotoxin E

OBJECTIVE To clone the gene of staphylococcal enterotoxins E and obtain recombinant protein(rSEE),and study the activities of rSEE on mice lymphocyte and its lethal effects on tumor cells.METHODS SEE Gene was obtained from the genome of Staphylococcus aureus FRI326 by PCR amplification.The correct fragment was subcloned into pGEX-4T-1 to construct an expression plasmid.Through glutathione Sepharose 4B affinity column and after the digestion with thrombin,the rSEE protein was purified.To ob- serve the activities of rSEE on mice lymphocyte and the lethal effects on tumor cells of the activated mice lymphocyte,in vitro culture system was utilized.RESULTS An expression plasmid of pGEX-4T-1-SEE was constructed,and purified rSEE was obtained.MTT results indicated that rSEE had strong ability to stimulate mice lymphocyte proliferation,which was a dose-dependent effect.With the proliferation of mice splenic lymphocyte,rSEE had a strong lethal effect on tumor cells K562-AD,K562 and B16.CONCLUSION Purified rSEE with a superantigen activity would provide bedrock for the study on anti-tumor mechanism of rSEE and construction of tar- geted anti-tumor fusion protein.