重组分枝杆菌CFP10ESAT6融合蛋白和CFP32的表达及其抗原性

摘要/Abstract

摘要： 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。

关键词: 分枝杆菌, 结核, 重组融合蛋白质类, 细菌蛋白质类

Abstract: Objective To clone and express recombinant CFP10-ESAT6 fusion protein and CFP32 protein ofMycobacterium , and analyze their antigenicity.  Methods The cfp10-esat6 fusion gene fromMycobacterium tuberculosisstrain H37Rv was amplified by PCR. DNA fragment encoding CFP32 fromMycobacterium bovisBacillus 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 inE.coliBL21, 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 inE.coliBL21 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%.  Conclusion s Recombinant CFP10-ESAT6 fusion protein and CFP32 protein with good antigenicity were successfully obtained.

Key words: Mycobacterium tuberculosis, recombinant fusion proteins, bacterial proteins

中图分类号:

孙文霞, 谭云洪, 袁仕善, 谭笑, 夏燕, 陈婧. 重组分枝杆菌CFP10ESAT6融合蛋白和CFP32的表达及其抗原性[J]. 中国防痨杂志, 2010, 32(6): 22-15.

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