结核杆菌早期分泌性蛋白ESAT-6基因测序质粒及真核表达重组质粒的构建

目的 构建结核杆菌早期分泌性蛋白ESAT—6基因测序质粒及真核表达重组质粒，为结核病的诊断、重组疫苗应用和免疫效应检测打下基础。方法 以结核杆菌H37Rv株基因组DNA为模板，用PCR法对基因ESAT—6进行扩增，将扩增的产物连接于测序载体pUCm—T上，经测序反应确定无误，再将PCR反应产物经酶切后，克隆于真核表达载体pcDNA3．1( )上。结果 构建了结核杆菌基因ESAT—6的重组测序质粒，并对其进行了测序。真核表达重组质粒pcDNA3．1( )—ESAT—6亦构建成功。结论 结核杆菌早期分泌性蛋白ESAT—6真核表达重组质粒的成功构建为结核病的诊断、重组疫苗应用和免疫效应检测以及相应抗原、抗体的制备打下基础。

Construction of sequencing recombinant plasmid and eukaryotic expression recombinant plasmid of ESAT-6 mycobacterium tuberculosis

Objective:To construct eukaryotic expression vector of ESAT-6 and to establish a foundation for diagnosis of tuberculosis,applying tuberculosis vaccine into clinic practice and detecting of immune effect.Methods:The gene encoding protein ESAT-6 was amplified from M.tuberculosis H37Rv genomic DNA by PCR technique. PCR product was cloned into sequencing vector pUCm-T. Sequence of ESAT-6 gene was proved by sequencing. After ESAT-6 gene was digested with BamH Ⅰ and EcoR Ⅰ, ESAT-6 gene was ligated to eukaryotic expression vector pcDNA3.1(+).Results:Sequencing vector of ESAT-6 gene was successfully constructed. Sequence of ESAT-6 gene was proved correct by sequencing. ESAT-6 gene was successfully inserted into the eukaryotic expression vector pcDNA3.1 (+).Conclusions:Recombinant pcDNA3.1 (+) ESAT-6 was obtained. It will establish the foundation for detecting of immunological effect and preparation of antigen and antibody of Ⅰ ESAT-6 protein on a large scale.