结核分枝杆菌Ag85B-Esat6-HspX融合基因真核表达载体的构建

摘要

目的 构建结核分枝杆菌Ag85B-Esat6-HspX融合基因,并对其在体外真核细胞中进行表达.方法 用PCR法从结核分枝杆菌H37Rv株基因组中分别扩增Ag85B、Esat6、HspX基因,插入到pUC19一T载体,序列测定正确后,将融合基因再次克隆到真核表达载体pcDNA3.1(-).重组质粒经酶切鉴定并测序正确后,用MegaTran1.0转染293T细胞,并用Western-Blot检测目的蛋白的表达.结果 Western-blot检测到分子量大小约65 kDa的目的蛋白.结论 成功地构建了结核分枝杆菌Ag85B-Esat6-HspX融合基因的真核表达载体,且该重组载体可在体外真核细胞中获得特异性的表达.%Objective To construct fused gene Ag85B-Esat6-HspX of Mycobacterium tuberculosis and investigate its expression in eukaryotic cells in vitro. Methods The gene encoding Ag85B, ESAT6 and HspX protein was amplified by PCR from genome of Mycobacterium tuberculosis H37Rv strain, and was inserted into cloning vector pUC19-T. After the sequence was confirmed ,the fused gene was subcloned to eukaryotic expression vector pcDNA3.1 ( - ). After identified by restriction enzymec digestion and verified by DNA sequencing again, the recombinant plasmid pcDNA-Ag85B-Esat6-HspX was transfected into 293T cells with MegaTran 1.0. Western blot was used to detect the expression of goal protein.Result A 65 kDa protein was detected with specific antibodies. Conclusion The eukaryotic expression vector pcDNAAgSSB-Esat6-HspX has been constructed successfully and the fusion protein could be expressed specifically in 293T cells.