结核分枝杆菌Mce2R/Rv0586蛋白的克隆、表达与纯化

Cloning,Expression and Purification of Mce2R/Rv0586 Protein from Mycobacterium Tuberculosis

构建pET-32m-Mce2R原核表达质粒,对重组蛋白Mce2R进行诱导表达、纯化及活性鉴定.以结核分枝杆菌H37Rv全基因组为模板,经聚合酶链式反应(PCR)扩增Mce2R(Rv0586)基因片段,产物经BamHⅠ和HindⅢ限制性内切酶酶切,电泳验证后与同样双酶切后的载体质粒pET32m进行连接,将连接产物pET-32m-Mce2R转化到大肠杆菌DH5a中,抽提质粒后测序验证,重组载体转化至大肠杆菌BL21(DE3)中以IPTG诱导表达,利用亲和层析纯化,经SDS-PAGE检测目的蛋白,分子量约为29 kDa.为进一步探索结核分枝杆菌Mce2R蛋白的结构及生化机制奠定了基础.

The prokaryotic expression plasmid pET-32m-Mce2R was constructed,and the recombinant protein Mce2R was induced,expressed,purified and identified.In this study,the whole genome of M.tuberculosis H37Rv was used as a template to amplify the Rv0586 gene fragment by polymerase chain reaction(PCR).The product was digested with two restriction enzymes,BamHⅠand HindⅢ.The cut vector plasmid pET32m was ligated with the enzyme-digested product,and the ligation product pET-32m-Rv0586 was transformed into E.coli DH5a.After plasmid extraction and sequencing verification,the recombinant vector was transformed into E.coli BL21(DE3)and the expression of the recombinant protein was induced by IPTG.After purification,the target protein was detected by SDS-PAGE with a molecular weight of about 29 kDa.This study has laid a foundation for further exploring the structure and biochemical mechanism of M.tuberculosis Mce2R protein.