结核分枝杆菌包涵体蛋白Rv3480c的克隆、表达与纯化

Cloning,expression and purification of the Rv3480c inclusion body protein of Mycobaterium tuberculosis

目的应用分子生物学技术对结核分枝杆菌重组抗原Rv3480c基因进行克隆、表达和纯化。方法以结核分枝杆菌标准菌株H37Rv基因组为模板,通过聚合酶链式反应(PCR)扩增,克隆Rv3480c基因片段,产物经验证后与载体质粒p ET28a连接重组质粒,将验证构建成功的重组质粒转化入表达宿主E.coli BL21(DE3)菌体内,经异丙基半乳糖苷(IPTG)诱导蛋白表达,溶解包涵体,并通过多次低温冻融、不同浓度尿素复性方法进行包涵体蛋白质的纯化。结果扩增后基因电泳试验及测序证实成功重组目的基因,用尿素溶液和磷酸盐缓冲液(PBS)透析和纯化重组蛋白,Western-Blot证实成功重组结核分枝杆菌抗原Rv3480c。结论应用分子生物学技术成功获得结核分枝杆菌Rv3480c蛋白,为其结构和功能的进一步研究奠定了基础,并为包涵体蛋白的纯化提供了新的技术思路。

Objective To clone,express and purify the Rv3480c inclusion body protein of Mycobaterium tuberculosis by molecular biological technique. Methods Rv3480c was amplified from M. tuberculosis H37Rv by PCR and cloned into vector pET-28a,then transformed to E. coli BL21（DE3） strain. The engineering bacteria were induced to express the protein by Isopropyl β-D-1-Thiogalactopyranoside（ IPTG）. The inclusion body was repeatedly dissolved,and recombinant protein was purified. Results The gene that was amplified by PCR was successfully built in right sequence detected via agarose gel electrophoresis and sequencing. Recombinant protein was dialyzed and purified with urea solution and Phosphate Buffered Saline（ PBS）. The target protein was identified as Rv3480c by Western-blot,which was the recombination antigen of M. tuberculosis. Conclusion The purified Rv3480c protein was successfully obtained by molecular biological technique,which provides an experimental basis for further studies of the function and structure of Rv3480c,and explores a new method for better purification of inclusion body proteins.