结核分枝杆菌CFP10-ESAT6融合蛋白在大肠杆菌中的高效表达

目的　在大肠杆菌中高效融合表达结核分枝杆菌分泌蛋白ESAT 6和CFP 10 ,获得纯化的重组ESAT6 CFP10 (rCFP10 ESAT6 )融合蛋白抗原。方法 通过聚合酶链反应 (polymerasechainreaction ,PCR)扩增CFP10 ESAT6融合基因 ;以质粒pET 2 8a为表达载体 ,构建重组质粒 ,转化大肠杆菌BL2 1(DE3) ;以异丙基硫代半乳糖苷 (IPTG)诱导表达目的蛋白 ,通过SDS PAGE电泳和蛋白免疫印迹法 (Westernblotting)鉴定rCFP10 ESAT6在大肠杆菌中的表达 ,确定rCFP10 ESAT6蛋白抗原在大肠杆菌中的表达形式 ;采用ChelatingSepharoseFastFlow蛋白纯化试剂纯化重组蛋白。West ernblotting及酶联免疫吸附试验 (ELISA)分析重组蛋白的免疫原性。结果 重组质粒pET2 8a CFP10 ESAT6中目的基因测序结果与报道序列相同 ;在大肠杆菌中以可溶性形式表达 ;分子量约2 8kDa ,表达量约占菌体总蛋白的 4 6 % ,纯化后的rCFP10 ESAT6样品经SDS PAGE和激光密度扫描分析表明其纯度为 90 %左右 ,每 10 0ml培养菌可获得 16mg左右的重组蛋白 ;Western印迹结果证实重组蛋白与His·tag单克隆抗体及确诊的肺结核病患者血清发生特异免疫反应。ELISA结果分析表明 ,该重组抗原能区分肺结核患者血清及正常人血清。结论 成功地表达和纯化了结核分枝杆菌CFP10-ESAT6融合蛋白。该重组蛋白具有特异的免疫原性。

Fused expression of Mycobacterium tuberculosis CFP-10 and ESAT-6 protein in Ecoli

Objective To obtain the recombinant CFP10-ESAT6 fused protein of M.tuberculosis .Methods The gene coding CFP-10 and ESAT-6 protein was amplified by polymerase chain reaction (PCR).The gene was inserted into an expression vector pET-28a to get recombinant plasmid.The recombinant plasmid was transformed into E.coli BL21(DE3).The E.coli carrying recombinant plasmid were induced by IPTG.The expressed product was indentified by SDS-PAGE and purified by metal chelation chromatography,and its immunological characteristics were analyzed by Western-blotting and ELISA technology.Results The sequence of CFP-10 and ESAT-6 in recombinant plasmid was consistented to that of Genbank report. The recombinant CFP10-ESAT6 protein was highly expressed in the form of solution in E.coli ,which was confirmed by Western-blotting analysis with monoclonal antibody against 6×His·tag and TB patient serum .ELISA test Results indicated that the purified recombinant protein could distinguish sera from tuberculosis patients and those from healthy people.Conclusion The recombinant CFP10-ESAT6 protein was expressed and purified successfully in the form of solution in E.coli and showed specific immunogenicity.