重组结核分枝杆菌特异性分泌抗原融合蛋白的活性鉴定及初步应用

目的：融合表达结核分枝杆菌分泌蛋白CFP10-ESAT-6或ESAT-6-CFP10，研究其免疫学特性，为结核病的血清学诊断提供物质基础。方法：将一个柔性的氨基酸“接头”插入原核表达载体pET32c（＋）中，构建pET32c（＋）-linker。PCR法扩增CFP10、ESAT-6基因。将CFP10克隆入改建的载体pET32e（＋）的linker前，ESAT-6克隆入linker后，构建CFP10-ESAT-6融合基因；或将ESAT-6克隆入改建的载体pET32c（＋）的linker前，CFP10克隆入linker后，构建ESAT-6-CFP10融合基因，分别转化大肠杆菌XL1-blue，抽提质粒，酶切鉴定；在大肠杆菌B121中表达，通过Western blot分析其抗原性。结果：两个目的基因分别被按顺序成功克隆入载体pET32c（＋）的linker前或后。重组质粒pET32c（＋）-CFP10-ESAT-6或pET32c（＋）-ESAT-6-CFP10靶基因的测序结果与预计序列完全一致。融合蛋白在B121菌中高效表达。Western blot分析表明，融合蛋白与活动性肺结核患者血清能发生特异性免疫反应。结论：成功地构建了多抗原基因DNA质粒；pET32e（＋）-CFP10-ESAT-6或pET32c（＋）-ESAT-6-CFP10质粒在B121菌中能高效表达rCFP10-ESAT-6或rESAT-6-CFP10融合蛋白，该蛋白兼具CFP10和ESAT-6两种蛋白的抗原性。本研究为rCFP10-ESAT-6或rESAT-6-CFP10融合蛋白在结核病血清学诊断中应用奠定了基础。

Molecular cloning, fused expression and characterization of Mycobacterium tuberculosis recombinant CFP10 -ESAT -6 or EAST -6 -CFP10 fusion protein

Objective:To investigate the fused expression of secreted protein CFP10-ESAT-6 or EAST-6-CFP10 of Mycobacterium tuberculosis and study their immunological characteristics and potential for serodiagnosis of tuberculosis.Methods:A pair of oligodeoxynucleotide named linker encoding 12 glycines and 3 serines was synthesized and cloned into plasmid pET32c(+) to construct pET32c(+)-linker.The CFP10 and ESAT-6 gene were amplified by PCR reaction and cloned into pET32c(+)-linker either ahead of or following linker.The recombinant CFP10-ESAT-6 or EAST-6-CFP10 fusion protein was expressed in E.coli.BL21.Their antigenicity were confirmed by Western blot.To evaluate the potential value of fusion proteins in the diagnosis of tuberculosis with sera of tuberculosis patients.Results:The sequence of recombinant plasmid pET32c(+)-CFP10-ESAT-6 or pET32c(+)-EAST-6-CFP10 was identical to the predicted sequence.The recombinant protein(rCFP10-ESAT-6 or rEAST-6-CFP10),about 26 000,existed in the cytoplasm BL21.Western blot showed that the rCFP10-ESAT-6 or rEAST-6-CFP10 had good immuoreactivity with sera from patients with active tuberculosis.Conclusion:Multi-antigen plasmid encoding CFP10 and ESAT-6 of Mycobacterium tuberculosis is constructed successfully.The fusion protein CFP10-ESAT-6 or EAST-6-CFP10 is obtained,so that it can provide an experimental basis for diagnosis of Mycobacterium tuberculosis in serology.