结核分枝杆菌CFP-10与Rv2626c蛋白的表达及血清学诊断研究

Expression and application of CFP-10 and Rv2626c proteins of Mycobacterium tuberculosis

目的通过构建结核分枝杆菌(结核菌)CFP-10和Rv2626c蛋白表达载体,并在大肠杆菌表达,对其免疫反应性进行鉴定评价。方法用PCR法从结核菌H37Rv基因组DNA分别扩增出CFP-10、Rv2626c基因,连接到表达载体PET30a上,在大肠杆菌中表达;组氨酸标签(His-Tag)镍柱层析纯化重组蛋白;用ELISA方法进行检测。结果构建了含CFP-10、Rv2626c重组质粒的大肠杆菌工程菌,发现目的蛋白主要以可溶形式存在;用重组CFP-10、Rv2626c蛋白组成联合抗原,ELISA方法检测214份血清,阳性率达77.1%。结论目的基因克隆入宿主菌中并成功表达,重组的CFP-10、Rv2626c蛋白组成联合抗原可能成为结核病血清学诊断的组合抗原之一。

In order to evaluate the potential of the antigens in serodiagnosis of TB,CFP-10 and Rv2626c genes were cloned and their proteins were expressed in Escherichia coli（E.coli）.The gene encoding CFP-10 and Rv2626c proteins were amplified by PCR from genome of M.tuberculosis H37Rv,and then inserted into expression vector PET30a and expressed fusion proteins CFP-10 and Rv2626c in E.coli BL21（DE3）.The two recombinant proteins were purified by affinity chromatography and detected by ELISA.Results showed that the two target proteins were expressed in E.coli after induction with IPTG.The solubility analysis indicated that the two recombinant proteins existed as soluble protein.And ELISA results showed the sensitivities of recombinant CFP-10,Rv2626c protein and CFP-10 combined with Rv2626c were 75.4%,44.1% and 77.1%,respectively.The specificities of the antibodies to CFP-10,Rv2626c and CFP-10 with Rv2626c were 85.4%,75.0 and 82.3%,respectively.There was no significant difference between the reactive rates of smear positive and negative TB patients.The gene sequences of CFP-10 and Rv2626c were obtained and the antigens were expressed in E.coli BL21.The results suggest that the CFP-10 protein combined with Rv2626c protein is a potential candidate as diagnostic reagent for the early detection of TB infection.