Rv2031c、38kDa及融合蛋白CFP10-ESAT6用于结核病血清学诊断的评价

Expression and preliminary evaluation of the recombinant antigens RV2031c and 38kDa and the recombinant chimeric antigen CFP10-ESAT6 in the serological diagnosis of tuberculosis

目的对Rv2031c、38kDa以及融合蛋白CFP10-ESAT6三种结核分枝杆菌抗原和抗原的混合物进行检测效能评价,筛选最佳抗原并分析其应用价值。方法以结核分枝杆菌标准株H37Rv菌株基因组为模板,PCR扩增目的基因;利用pET-30a、pET-32a载体构建重组质粒,在宿主E.coli BL21(DE3)细胞中诱导表达,利用His标签亲和镍柱纯化重组蛋白。分别用单种重组蛋白和几种蛋白混合后包被酶标板,采用ELISA对129份血清(39份结核病患者血清和90份健康者血清)进行检测。结合受试者工作特征曲线法计算不同抗原和混合抗原的最佳临界值(Cut-off value)包被浓度,得出检测的灵敏度、特异度和曲线下面积,对其诊断效能和应用价值进行综合评价。结果以Rv2031c、38kDa和CFP10-ESAT6为包被抗原的ELISA法检测相应抗体的灵敏度分别为41.0%,51.3%和46.2%,特异度分别为90.0%、88.9%和79.1%,曲线下面积分别为0.640、0.761和0.690,以3种蛋白混合物为包被抗原的3项指标分别为66.7%、77.8%和0.746。结论 Rv2031c、38kDa以及融合蛋白CFP10-ESAT6 3种抗原混合物的诊断效能较好,具有一定的临床应用价值。

Objective To evaluate the effectiveness of the recombinant antigens Rv2031c and 38kDa and the recombi- nant chimeric antigen CFP10-ESAT6 of Mycobacterium tuberculosis and a mixture of those antigens to serologically diag- nose tuberculosis. Methods PCR was used to amplify the three antigens from the genomic DNA of the H37Rv strain of M. tuberculosis. The product was cloned into pET-30a and pET32a vectors and transformed into E. coli BL21 （DE3） strain for expression. Proteins were purified using Ni-NTA His-binding resin affinity chromatography according to the manufacturer＇s protocol. Three single proteins and a mixture of those proteins were coated on microtiter plates. The three recombinant antigens were used to perform an enzyme-linked immunosorbent assay （ELISA） on 129 sera samples, inclu- ding 39 from patients with TB and 90 from healthy people. The receiver operating characteristic （ROC） curve was plotted to calculate the cut-off value for antigen titers, and sensitivity, specificity, the area under the curve （AUC）, and diagnos- tic efficiency were then calculated. The three antigens and a mixture of those antigens were evaluated for their ability to serologically diagnose tuberculosis. Results ELISA with Rv2031 as a coating antigen detected antibodies against the an- tigen with a sensitivity of 41.0%, a specificity of 90.0%, and an area under the curve （AUC） of 0. 640. ELISA with 38 kDa as a coating antigen detected antibodies against the antigen with a sensitivity of 51.3%, a specificity of 88.9%, and an AUC of 0. 761. ELISA with CFP10-ESAT6 as a coating antigen detected antibodies against the antigen with a sensitivity of 46.2%, a specificity of 79.1%, and an AUC of 0. 690. ELISA with a mixture of the three antigens detected anti- bodies against the antigens with a sensitivity of 66.7%, a specificity of 77.8%, and an AUC of 0. 746. Conclusion A mixture of the antigens Rv2031c, 38 kDa, and CFP10-ESAT6 has better diagnostic efficiency than each antigen alone, and the mixture has clinical utility to some extent.