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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。
Objective To construct prokaryotic expression vector of Culture filtrate protein 21 (CFP21) of Mycobacterium tuberculosis and obtain CFP21 protein. Methods The genomic DNA of Mycobacterium tuberculosis strain H37Rv was used as a template to amplify the cfp21 gene by PCR. The plasmid pET28a (+) was used as the expression vector to construct the plasmid pET28a (+) / cfp21. The plasmid was transformed into Escherichia coli DH5α, PCR was amplified, sequenced and transformed into Escherichia coli BL21. The expression was induced by IPTG, analyzed by SDS-PAGE, and CFP21 was purified by His Bind protein purification kit. Results The CFP21 was constructed, expressed and purified. The molecular weight of CFP21 was about 24kD, which mainly existed as inclusion body. Conclusion The target gene was cloned into host bacteria and expressed successfully. CFP21 protein was purified, which laid the foundation for the further study of CFP21.