结核分枝杆菌Ag85蛋白复合物的原核表达及其在血清学诊断中的应用研究

Prokaryotic expression of Mycobacterium tuberculosis Ag85 protein complexes and a preliminary study on the serological diagnosis of tuberculosis

目的构建结核分枝杆菌Ag85复合物原核表达载体并表达和纯化重组蛋白,评价该家族蛋白在结核病血清学诊断中的应用价值。方法以标准株H37Rv基因组为PCR模板扩增fbpA、fbpB及fbpC基因并克隆至原核表达载体,转化至C43(DE3)菌株后经诱导、表达、纯化获得重组蛋白Ag85A、Ag85B、Ag85C。分别以纯化蛋白包被酶标板,采用方阵滴定确定Ag85A、Ag85B、Ag85C蛋白间接ELISA的最佳条件,用优化的ELISA检测367份健康对照组血清及86份痰涂阳性结核病患者血清中的特异IgM、IgG抗体。结果成功构建Ag85复合物基因的重组质粒,经诱导表达获得Ag85A、Ag85B、Ag85C重组蛋白。分别以纯化的Ag85A、Ag85B、Ag85C为包被抗原,采用ELISA检测结核病人血清IgG抗体,灵敏度分别为65.1%、69.8%和41.8%,特异性分别为75.1%、78.1%和64.2%;以Ag85B+Ag85A或Ag85CIgG抗体阳性为判断标准,检测血清结核分枝杆菌IgG抗体的灵敏度为60.4%,特异性为86.1%。结论以Ag85B+Ag85A或Ag85C血清IgG抗体阳性为判断标准,检测血清结核分枝杆菌IgG抗体具有较好的灵敏度与特异性,可作为结核病血清学诊断方法。

Objectives To construct a prokaryotic expression vector for expression of Mycobacterium tuberculosis Ag85 complexes,to express and purify those proteins,to evaluate the value of those proteins in detecting serum antibodies in patients with tuberculosis,and to provide a theoretical basis for rapid diagnosis of tuberculosis. Methods The fbpA,fbpB,and fbpCgenes were amplified from the H37 Rv genome and cloned into a prokaryotic expression vector.The recombinant plasmids were transformed into the C43（DE3）strain.Protein expression was induced and the resulting proteins were purified,yielding the recombinant proteins Ag85 A,Ag85 B,and Ag85 C.Optimal conditions for indirect ELISA of the proteins Ag85 A,Ag85 B,and Ag85 Cwere determined using square titration.IgM and IgG antibodies were detected in sera from 367 healthy controls and 86 patients with sputum smears that were positive for tuberculosis using indirect ELISA. Results Recombinant plasmids for expression of the Ag85 complex were successfully constructed,the proteins were purified,and the proteins Ag85 A,Ag85 B,and Ag85 C were obtained.The optimal antigen concentration for ELISA was 0.25μg/ml（Ag85 A）and 0.1μg/ml（Ag85 Band Ag85 C）.The optimal serum dilution was 1：100（Ag85 A）and 1：50（Ag85 Band Ag85 C）.For IgM,the dilution factor for the secondary antibody was 1：8 000（Ag85 A）and 1：12 000（Ag85 Band Ag85 C）.For IgG,the optimal dilution factor for the secondary antibody was 1：20 000（Ag85 Aand Ag85 B）and 1：40 000（Ag85 C）.Ag85 Adetected IgG-positive serum with a sensitivity of 65.1%,Ag85 B detected it with a sensitivity of 69.8%,and Ag85 Cdetected it with a sensitivity of 41.8%.Ag85 Adetected IgG-positive serum with a specificity of 75.1%,Ag85 Bdetected it with a sensitivity of 78.1%,and Ag85 Cdetected it with a sensitivity of 64.2%.With positivity for the Ag85 Aand Ag85 Bantigens and IgG antibodies or positivity for the Ag85 Cantigen and IgG antibodies as a standard,serum IgG antibodies had a sensitivity of 60.4%and a specificity of 86.1%in detecting M.tuberculosis. Conclusion With positivity for the Ag85 Aand Ag85 Bantigens or the Ag85 Cantigen in serum with IgG antibodies as a standard,serum IgG antibodies had better sensitivity and specificity at detecting M.tuberculosis and can provide a basis for the rapid diagnosis of tuberculosis.