结核分枝杆菌rESAT-6-CFP-10融合蛋白的构建和表达及其免疫反应性分析

Objective To construct esat-6-cfp-10 fusion gene (ec) and its expression vector pET-23b( + )-esat-6-cfp-10 pET-23b( + )-ec], and express rESAT-6-CFP-10 (rEC)fusion protein of Mycobacterium tuberculosis (MTB) H37Rv and evaluate its immunoreactivity. Methods Fused gene encoding ESAT-6 and OP-10 of MTB was linked with the (Gly4Ser)3 linker by gene SOEing. Construction of TA cloning vector pMDR 19-T-esal-6-cfp-10 (pMDR 19-T-ec) was identified by PCR, restriction analysis and sequencing. After pMDR 19-T-ec was digested with double restriction enzymes (Nde Ⅰ and Xho Ⅰ ), the fused gene was inserted into prokaryotic expression vector pET-23b ( + ). The recombinant plasmid pET-23b( + )-ec was transformed into E. coli BL21(DE3) pLysE, and IPTG was used to induce the expreestion of rEC. Its expression was detected with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. rEC was purified by nickel-chelate affinity chromatography. The immunoreactivity of rEC was preliminarily evaluated by Western blot. Results A recombinant plasmid pET-23b( + )-ec was successfully constructed and could stably express a 21 000 rEC, which accounted for 35% total proteins of bacillus. It existed in soluble form in E. coli. After purification, the purity of rEC reached 97.2% . Its immunoreactivity was confirmed by Western blot. Conclusions The prokaryotic expression vector pET-23b( + )-ec has been constructed and the fusion protein rEC has been obtained successfully, which provides an experimental basis for the application of rEC in the diagnosis of tuberculosis.

Study on construction and expression and immunoreactivity of Mycobacterium tuberculosis recombinant ESAT-6-CFP-10