结核分枝杆菌重组双歧杆菌-ESAT-6-MPT64疫苗的构建及鉴定

The Construction and Identification of Recombinant Bb-ESAT-6-MPT64 Vaccine of Mycobacterium Tuberculosis

目的构建并鉴定结核分枝杆菌重组双歧杆菌((Bifidobacteria bifidum,Bb)-ESAT-6-MPT64疫苗。方法以结核分枝杆菌H37Rv标准株基因组DNA为模板,通过PCR扩增ESAT-6和MPT64单基因序列,然后采用基因拼接(gene SOEing)法构建融合基因ESAT-6-MPT64,定向克隆至穿梭表达载体pGEX-1λT中,构建重组质粒pGEX-ESAT-6-MPT64;再电转化入Bb,构建结核分枝杆菌重组双歧杆菌-ESAT-6-MPT64疫苗。结果 PCR成功扩增出1020bp的ESAT-6-MPT64融合基因;双酶切证实ESAT-6-MPT64融合基因成功插入pGEX-1λT中;PCR证实rBb-ESAT-6-MPT64疫苗构建成功。结论成功构建了结核分枝杆菌rBb-ESAT-6-MPT64疫苗,为进一步研究其免疫保护效果奠定了基础。

Objective To construct and identify recombinant Bb-ESAT-6-MPT64 vaccine of Mycobacterium tuberculosis.Methods ESAT-6 and MPT64 antigen gene were amplified by PCR from standard genomic DNA of Mycobacterium tuberculosis H37Rv,and the constructed fusion gene ESAT-6-MPT64 by gene SOEing was cloned into plasmid pGEX-1λT to construct pGEX-ESAT-6-MPT64.The recombinant plasmid was electroporated into Bifidobacteria bifidum（Bb） to construct rBb-ESAT-6-MPT64 vaccine.Results 1020bp fusion gene of ESAT-6-MPT64 was successfully amplified by PCR and cloned into pGEX-1λT by restriction endonuclease analysis,rBb-ESAT-6-MPT64 vaccine was successfully constructed by PCR.Conclusion rBb-ESAT-6-MPT64 vaccine of Mycobacterium tuberculosis is successfully constructed,which may provide basis for the immunization of new anti-tuberculosis vaccine.