人结核杆菌热休克蛋白65和Ag85A基因真核双表达质粒的构建和体外表达

Construction and expression of eukaryotic co-expression plasmid containing Mycobacterium tuberculosis Hsp65 and Ag85A genes

目的构建人结核杆菌热休克蛋白65(Hsp65)与Ag85A基因的共表达载体pIRES-Hsp65-Ag85A,并检测其在体外的表达。方法利用聚合酶链反应(PCR)法及基因重组技术,以人结核杆菌基因组DNA为模板,扩增获得Hsp65与Ag85A的全长基因,并将其构建到真核表达质粒pIRES中获得共表达质粒pIRES-Hsp65-Ag85A。将该质粒转染Hela细胞,通过RT-PCR的方法检测目的基因的表达。结果经过测序证实重组质粒构建成功,该质粒在体外转染Hela细胞后可表达Hsp65与Ag85A两者的mRNA。结论成功构建了共表达质粒pIRES-Hsp65-Ag85A,该质粒能在人子宫颈癌细胞中表达。

Objective To construct a eukaryotic co-expression plasmid containing Mycobacterium tuberculosis （MTB） Hsp65 and Ag85A genes. Methods Hsp65 and Ag85A genes were obtained by PCR and cloned into eukaryotic expression plasmid plRES to construct recombinant plasmid plRES-Hsp65 Ag85A. The recombinant plasmid was transfected into Hela cells and the expression of target genes was detected by RT-PCR. Results Restriction enzyme analysis, PCR and sequencing results showed that the recombinant plasmid plRES-Hsp65-Ag85A was constructed successfully and the expression of Hsp65 and Ag85A genes could be detected by RT-PCR. Conclusion The eukaryotic expression plasmid plRES-Hsp65-Ag85A containing Mycobacterium tuberculosis Hsp65 and Ag85A genes was constructed successfully and can co-express Hsp65 and Ag85A in vitro, which lays foundation for further development of DNA vaccine against tuberculosis.