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.