【作者】 薛乔； 应跃斌； 潘映秋； 李丹曦； 孙红颖； 陈枢青；

【Author】 Xue Qiao, Ying Yue-bing, Pan Ying-qiu, Li Dan-xi, Sun Hong-ying, CHEN Shu-qing (Department of Biopharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310031. P. R. China)

【机构】 浙江大学药学院生物制药研究室；

【摘要】 目的克隆金黄色葡萄球茼肠毒素SEC2全长基因,构建SEC2的表达戢体,实现其可溶性表达,并对纯化的rSEC2蛋白的生物学活性进行研究。方法通过聚合酶链式反应 (Polymerase chain reaction,PCR)从金黄色葡萄球菌FRI1230菌株基因中得到肠毒素SEC2 的基因,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素和层析纯化．通过重组SEC2对淋巴细胞的增殖作用及其对肿瘤细胞杀伤活性的影响,对其进行超抗原活性和免疫学活性进行研究。结果得到正确的肠毒素SEC2基因序列并得到高效表达的融合蛋白,MTT法结果表明重组SEC2表现出良好的促淋巴细胞增殖且能够增强淋巴细胞对肿瘤细胞的杀伤活性。结论本实验成功克隆了SEC2基因,表达并纯化出具有抗肿瘤生物学活性的重组 SEC2蛋白,为进一步对其分子抗肿瘤作用机制的研究,构建靶向抗肿瘤融合蛋白奠定了基础．更多还原

【Abstract】 Aim To clone the gene of Staphylococcal enterotoxins C2 and express it in the form of a soluble fusion protein in E.coli. Then SEC2 activation on mice lymphocyte and its lethal effects on tumor cells was studied. Methods Staphylococcus aureus SEC2 gene was cloned in to GST gene fusion vector pGEX-4T-1.The resultant plasmid pGEX-4T-SEC2 was used to transform E. coli BL21, where the GST-SEC2 fusion protein was expressed efficiently. The rSEC2 protein was purified with Glutathione Sepharose 4B affinity column and digestion with thrombin. We utilized the in vitro culture system to observing the activation of the SEC2 on mice lymphocyte and the lethal effects on tumor cells of the activated mice lymphocyte. Results The proper gene of SEC2 was cloned and we got the purified rSEC2. The MTT results indicated that rSEC2 have strong ability to stimulate mice lymphocyte to proliferate, which is a dose-dependent effect With the proliferation of mice splenic lymphocyte, rSEC2 has strong lethal effect on tumor cells,B16.K562 and K562-AD. Conclusion In mis study, we cloned the gene of SEC2 and the rSEC2 protein which had strong lethal effect on tumor cells B16,K562 and K562-AD was obtained.更多还原