结核分枝杆菌分泌蛋白Ag85B、Hsp16.3和ESAT6在血清学检测中的初步应用  

Application of Ag85B,Hsp16.3 and ESAT6 of Mycobacterium Tuberculosis in Serological Detection

作　　者：张彩勤[1] 张海[1] 赵勇[1] 毛峰峰[1] 赵善民[1] 杨巍[1] 白冰[1] 师长宏[1] 

机构地区：[1]第四军医大学实验动物中心,陕西西安710032

出　　处：《现代生物医学进展》2011年第3期401-404,共4页Progress in Modern Biomedicine

基　　金：国家"863"专项课题(2007AA02Z473);国家自然科学基金(30972767);陕西省自然科学基金(SJ08C203)联合资助

摘　　要：目的:探讨结核分枝杆菌分泌蛋白Hsp16.3、Ag85B以及融合蛋白ESAT6-CFP10、Ag85B-Hsp16.3和Ag85B-ESAT6用于TB病人血清学检测的意义。方法:将已构建的含5种目的基因的表达载体(pProEXHTb-Hsp16.3、pProEXHTa-Ag85B、pProEXHTb-ESAT6-CFP10、pProEXHTa-Ag85B-Hsp16.3、pProEXHTa-Ag85B-ESAT6),分别转入宿主菌E.coli DH5α中,诱导表达后分别获得Hsp16.3、Ag85B、ESAT6-CFP10、Ag85B-Hsp16.3和Ag85B-ESAT6五种蛋白,采用Ni2+亲和层析柱进行纯化,并用透析方法进行目的蛋白的复性。将经过复性的5种蛋白分别作为抗原,采用间接ELISA方法检测待测的血清样本,经OPD显色,测定各孔OD490值并判定结果。结果:五种蛋白被成功纯化并复性,通过ELISA方法共检测了22例TB病人血清、10例非结核病人血清和6例正常对照血清,Hsp16.3、Ag85B、ESAT6-CFP10、Ag85B-Hsp16.3和Ag85B-ESAT6这5种抗原的灵敏度分别为36.4%、90.9%、77.3%、95.5%、100%,特异性分别为100%、75%、100%、93.8%、93.8%。统计分析显示,ESAT6-CFP10和Ag85B、Ag85B-Hsp16.3、Ag85B-ESAT6这三种蛋白ELISA检测的结果无差异,而与Hsp16.3和痰涂片检测结果有显著差异。结论:Ag85B-Hsp16.3和Ag85B-ESAT6可作为结核分枝杆菌ELISA检测的初选抗原。Objective：To investigate the significance of Hsp16.3,Ag85B,ESAT6-CFP10,Ag85B-Hsp16.3 and Ag85B-ESAT6 of Mycobacterium tuberculosis（MTB） in the serological detection.Methods：The five recombinant plasmids（pProEXHTb-Hsp16.3,pProEXHTa-Ag85B,pProEXHTb-ESAT6-CFP10,pProEXHTa-Ag85B-Hsp16.3 and pProEXHTa-Ag85B-ESAT6） were transfered into E.coli DH5α,respectively.The five proteins were induced by IPTG and purified by Ni-NTA purification system under denaturing condi-tion.Following by renaturation of dialysis,the five proteins were used as antigen to detect sera of 22 TB patients,10 non-TB patients and 6 controls by indirect ELISA.OPD was chromogenic agent,and OD490 were detected.Results：The sensitivities of Hsp16.3,Ag85B,ESAT6-CFP10,Ag85B-Hsp16.3 and Ag85B-ESAT6 to 22 TB patients were 36.4 %,90.9 %,77.3 %,95.5 %,100%,respectively,while the specificity to 16 non-TB patients were 100 %,75 %,100 %,93.8 %,93.8 %,respectively.There was no difference for the ELISA detec-tion result between ESAT6-CFP10 and Ag85B,Ag85B-Hsp16.3,Ag85B-ESAT6,while there was difference between ESAT6-CFP10 with Hsp16.3 and sputum-smear.Conclusion：Ag85B-Hsp16.3 and Ag85B-ESAT6 could be used as the original selective antigens in de-tecting MTB by ELISA.

关 键 词：结核分枝杆菌 AG85B HSP16.3 ESAT6 酶联免疫吸附测定 

分 类 号：R378.911[医药卫生—病原生物学] Q75[医药卫生—基础医学]