重组分枝杆菌CFP10-ESAT6融合蛋白和CFP32的表达及其抗原性

目的 重组表达结核分枝杆菌CFP10和ESAT 6的融合蛋白及卡介苗（BCG）CFP32，分析其抗原性。 方法 用重组PCR从结核分枝杆菌标准株H37Rv基因组DNA中扩增获得融合基因cfp10-esat6；从BCG基因组DNA中扩增cfp32基因。经克隆和测序分析后，分别亚克隆至表达载体pQE-30和pET-23a(+)，在大肠埃希菌BL21中表达重组蛋白，予以纯化、 鉴定，Western blot和间接ELISA分析重组蛋白的抗原性。 结果构建了重组表达质粒pQE30-cfp10-esat6和 pET-cfp32，表达了CFP10-ESAT6融合蛋白和CFP32。CFP10-ESAT6蛋白表达量约占菌体总蛋白的15.2%，纯化后蛋白纯度约为92%，浓度为0.456-g/L；CFP32蛋白表达量约占菌体总蛋白的10.6%，纯化后蛋白纯度约为90%，浓度为0.310-g/L。纯化CFP10-ESAT6融合蛋白和CFP32检测结核病的敏感性分别为85.7%和71.4%，特异性均为100%。 结论 重组表达获得具有良好抗原性的重组融合蛋白CFP10-ESAT6和重组蛋白CFP32。

Clone and expression of CFP10-ESAT6 fusion protein and CFP32 protein of Mycobacterium and their antigenicity

Objective To clone and express recombinant CFP10-ESAT6 fusion protein and CFP32 protein of Mycobacterium,and analyze their antigenicity. Methods The cfp10-esat6 fusion gene from Mycobacterium tuberculosis strain H37Rv was amplified by PCR. DNA fragment encoding CFP32 from Mycobacterium bovis Bacillus Calmette-Guerin（BCG） was obtained by PCR. After cloning and sequence analysis,the cfp10-esat6 fusion gene and cfp32 gene were subcloned into expression vector pQE-30 and pET-23a（＋）,respectively. The recombinant proteins were expressed in E.coli BL21,purified by affinity chromatography,and analyzed by SDS-PAGE. Their antigenicities were analyzed by Western blot and indirect ELISA. Results Recombinant expression plasmid pQE30-cfp10-esat6 and pET-cfp32 were constructed. The recombinant CFP10-ESAT6 fusion protein and CFP32 protein were expressed in E.coli BL21 and were purified by affinity chromatography. The purified CFP10-ESAT6 and CFP32 proteins were used to detect the antibodies against tuberculosis,their sensitivities were 85.7% and 71.4%,respectively,their specificities were all 100%. Conclusions Recombinant CFP10-ESAT6 fusion protein and CFP32 protein with good antigenicity were successfully obtained.