论文部分内容阅读
目的 为结核病新型疫苗研究提供靶基因和靶抗原。方法 采用PCR扩增的方法获得结核杆菌两种免疫保护性抗原Ag85A及ESAT - 6的基因 ,将其定向克隆入真核及原核穿梭表达型载体 pBK -CMV构建含嵌合目的基因的重组质粒 ,转化大肠杆菌后用IPTG进行诱导表达 ,并通过SDS -PAGE和Western -blotting对表达蛋白进行初步分析。结果 1)从结核杆菌H37Rv株基因组DNA中扩增出Ag85A及ESAT - 6基因。 2 )成功构建了结核杆菌Ag85A及ESAT - 6双价抗原融合表达质粒 pBK - 85A -E6。 3)重组质粒 pBK - 85A -E6经IPTG诱导后能在大肠杆菌中稳定表达 38kDa的融合蛋白。 结论 成功构建了结核杆菌Ag85A及ESAT - 6双价抗原融合表达载体 ,并在大肠杆菌中实现了稳定表达 ,为进一步研究其在结核病基因工程疫苗研制中的应用奠定了基础。
Objective To provide target genes and target antigens for novel vaccine research on tuberculosis. Methods The genes of two immunoprotective antigens Ag85A and ESAT - 6 of Mycobacterium tuberculosis were obtained by PCR amplification and cloned into the eukaryotic and prokaryotic shuttle expression vector pBK - CMV to construct the recombinant plasmid containing chimeric gene. The recombinant plasmid was transformed into E.coli and induced by IPTG. The expressed protein was analyzed by SDS-PAGE and Western-blotting. Results 1) Ag85A and ESAT - 6 genes were amplified from the genomic DNA of Mycobacterium tuberculosis H37Rv strain. 2) Mycobacterium tuberculosis Ag85A and ESAT - 6 bivalent antigen fusion expression plasmid pBK - 85A - E6 were successfully constructed. 3) The recombinant plasmid pBK - 85A - E6 was induced by IPTG to express 38kDa fusion protein in E. coli stably. Conclusion The fusion expression vectors of Mycobacterium tuberculosis Ag85A and ESAT - 6 bivalent antigen were successfully constructed and stably expressed in E. coli, which laid the foundation for further study on its application in the development of genetic engineering vaccine for tuberculosis.