【作者】 朱中元； 张丹； 王海波； 肖劲逐； 邱一帆； 颜磊； 陈德； 刘爱国； 杨欣；

【Author】 Zhu Zhong-yuan,Zhang Dan,Wang Hai-bo,(Hainan Provincial Nongken Hospital1,Haikou,Hainan, China 570311;The College of Environment and Plant Protection of Hainan University2,Haikou,Hainan, China 570228,Nanjing Potomac BioTechnology Co LTD,Nanjing Jiangsu China 200800)

【机构】 海南省农垦总医院； 海南大学环境与植物保护学院； 南京大渊生物技术有限责任公司；

【摘要】 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CFP10、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CFP10、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表达成功,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。更多还原

【Abstract】 Objective 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.Methods The gene encoding CFPIO and Rv2626c proteins were amplified by PCR from genome of M.tuberculosis H37Rv,and then inserted into expression-vector PET30a and expressed fusion proteins CFPIO and Rv2626c in E.coli BL21(DE3).The two recombinant proteins were purified by affinity chromatography and detected by EL1SA.Results The two target proteins were expressed in E.coli after induction with IPTG.The solubility analysis showed that the two recombinant proteins existed as soluble protein.ELISA results showed the sensitivity of recombinant CFP-10 protein combined with recombinant Rv2626c protein was 77.1%.Conclusion The gene sequences of CFPIO and Rv2626c were obtained and the antigens were expressed in E.coli BL21.The results indicated that the CFP-10 protein combined with Rv2626c protein was a potential candidate as diagnostic reagent for the early detection of TB infection.更多还原