野生型金黄色葡萄球菌肠毒素C2在E.coli中的表达和纯化研究

目的克隆金黄色葡萄球菌肠毒素C2(SEC2)基因在E.coli重组表达,纯化重组SEC2(rSEC2)并进行生物学活性分析。方法PCR获得正确编码SEC2的基因片断,构建表达质粒pET-28a-sec2在E.coliBL21中表达。利用离子交换和分子筛色谱纯化rSEC2,四甲基偶氮唑盐(MTT)法对rSEC2和天然SEC2(nSEC2)生物学活性进行分析和比较。结果可溶性rSEC2占菌体总蛋白质的40%,两步纯化后蛋白质纯度约达95%,rSEC2和nSEC2均具有显著的超抗原活性。结论成功获得与nSEC2有着类似活性的高纯度rSEC2,为进一步研究该蛋白质的抗肿瘤机理奠定物质基础。

Research on the expression of recombinant wild-type staphylococcal enterotoxin C2 in E. Coli and its purification

PurposeTo construct an E.coli expression system for the staphylococcal enterotoxin C2(SEC2),purify it and analyze it′s bioactivity.MethodsThe sec2 gene with two endonucleases sites was amplified by PCR,then the segment was inserted into pET-28a(+) and the expression vector pET-28a-sec2 was constructed.The recombinant protein was induced in E.coli BL21 with IPTG and purified by anion-exchange chromatography and molecular exclusion chromatography.The bioactivity of both the rSEC2 and native SEC2(nSEC2) was analyzed by MTT method and the results were compared by t-test.ResultsThe nucleotides sequence was confirmed to code for the protein identical with nSEC2 except an excessive Met at N terminus,and the soluble rSEC2 was expressed to be 40% of total protein.The purity of the protein reached 95% after purification.The rSEC2 and nSEC2 were both proved to have typical superantigen activty.ConclusionSuccessfully obtained high purity rSEC2,which has identical superantigen activty with nSEC2,lay the foundation for further research on the mechanism of it′s anticancer activity.