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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 efficacy of three mixtures of Rv2031c, 38kDa and fusion protein CFP10-ESAT6 against Mycobacterium tuberculosis antigen, select the best antigen and analyze its value. Methods The target gene was amplified by PCR using the Mycobacterium tuberculosis H37Rv strain as a template. The recombinant plasmid was constructed by using pET-30a and pET-32a vectors and induced in E. coli BL21 (DE3) cells. Purification of Recombinant Proteins with Nickel and Nickel Columns. One hundred recombinant proteins and several proteins were mixed and coated with ELISA plates, and 129 serum samples (39 samples of tuberculosis and 90 healthy samples) were detected by ELISA. Combined with the receiver operating characteristic curve method, the optimal cut-off value of different antigens and mixed antigens was calculated, and the sensitivity, specificity and area under the curve were obtained. The diagnostic efficacy and application value Overview. Results The sensitivity of the corresponding antibodies detected by ELISA with Rv2031c, 38kDa and CFP10-ESAT6 as coating antigen were respectively 41.0%, 51.3% and 46.2%, and the specificity was 90.0%, 88.9% and 79.1% respectively. The areas under the curve were 0.640, 0.761 and 0.690 respectively. The three indexes of the three protein mixtures as coating antigen were 66.7%, 77.8% and 0.746, respectively. Conclusion The diagnostic efficiency of Rv2031c, 38kDa and fusion protein CFP10-ESAT6 is good and has certain clinical value.