结核分枝杆菌培养滤液蛋白21(CFP21)的基因克隆、表达及纯化

Cloning,expression and purification of the gene of antigen culture filtrate protein 21 of Mycobacterium tuberculosis

目的构建结核分枝杆菌培养滤液蛋白21(Culture filtrate protein 21,CFP21)原核表达载体,获得CFP21蛋白。方法以结核分枝杆菌H37Rv株基因组DNA为模板,PCR扩增cfp21基因,质粒PET28a(+)为表达载体,构建PET28a(+)/cfp21质粒,转化入大肠埃希菌DH5α中,抽提质粒,PCR扩增,测序,转化到大肠埃希菌BL21中,用IPTG诱导表达,SDS-PAGE分析,并用His Bind蛋白纯化试剂盒纯化CFP21。结果构建、表达和纯化了CFP21,分子量约为24kD,主要以包涵体形式存在。结论目的基因克隆入宿主菌中并表达成功,纯化得到CFP21蛋白,为CFP21的进一步研究奠定基础。

In order to construct culture filtrate protein 21（CFP21） vector of Mycobacterium tuberculosis（Mtb） and express it in E.coli BL21,the cfp21 gene was amplified from Mtb H37Rv genomic DNA using PCR in vitro,and inserted into vector PET-28a（＋） to construct the recombinant plasmid.The recombinant plasmid was then under the procedure of transforming into E.coli DH5α,plasmid extracting,PCR amplificating,sequencing,and transforming into E.coli BL21.The recombinant plasmid was induced with IPTG and identified using SDS-PAGE.Then the protein was purified using His Bind purification kit.Results demonstrated that CFP21 has been constructed,expressed and purified successfully.The molecular weight was about 24KD and the form of expression was inclusion bodies.In conclusion,the target gene has been cloned into host bacterium and expressed successfully.The purified recombinant protein CFP21 paves the way for TB diagnosis and vaccine development in the future.