结核杆菌Ag85A及ESAT-6双价抗原融合表达载体的构建及其在大肠杆菌中的高效表达

Construction the fused expression vector of Mycobacterium tuberculosis Ag85A and ESAT-6 antigens and its expression in E.coli

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。

Aim To determine the target genes and target antigens for developing new vaccine of tuberculosis Methods Two genes encoding Ag85A and ESAT-6 proteins of Mycobacterium tuberculosis H37Rv strain were amplified by using polymerase chain reaction (PCR) The two amplification products were inserted into plasmid vector pBK-CMV to construct recombinant plasmid which contains chimeric target genes The recombinant plasmid was transfered into E coli XLl-Blue MFR′and was expressed under the inducement of IPTG The expression product was analyzed by using SDS-PAGE and western-blotting Results 1) The two genes encoding Ag85A and ESAT-6 proteins of Mycobacterium tuberculosis were obtained by using PCR 2) A recombinant fused expression vector of Mycobacterium tuberculosis Ag85A and ESAT-6 antigen was constructed 3) The recombinant plasmid can stably express a 38kDa fusion protein in E coli XLl-Blue MRF′after induced with IPTG Conclusion A recombinant plasmid which can express Mycobacterium tuberculosis Ag85A and ESAT-6 fusion protein has been successfully constructed The recombinant plasmid can stably express 38kDa fusion protein in E coli XLl-Blue MFR′ These results provide the basis for the further the study the usefulness of the fusion protein and the recombinant plasmid in new vaccine against Mycobacterium tuberculosis