【摘要】：目的對Rv2031c、38kDa以及融合蛋白CFP10-ESAT6三種結(jié)核分枝桿菌抗原和抗原的混合物進(jìn)行檢測效能評價,篩選最佳抗原并分析其應(yīng)用價值。方法以結(jié)核分枝桿菌標(biāo)準(zhǔn)株H37Rv菌株基因組為模板,PCR擴(kuò)增目的基因;利用pET-30a、pET-32a載體構(gòu)建重組質(zhì)粒,在宿主E.coli BL21(DE3)細(xì)胞中誘導(dǎo)表達(dá),利用His標(biāo)簽親和鎳柱純化重組蛋白。分別用單種重組蛋白和幾種蛋白混合后包被酶標(biāo)板,采用ELISA對129份血清(39份結(jié)核病患者血清和90份健康者血清)進(jìn)行檢測。結(jié)合受試者工作特征曲線法計算不同抗原和混合抗原的最佳臨界值(Cut-off value)包被濃度,得出檢測的靈敏度、特異度和曲線下面積,對其診斷效能和應(yīng)用價值進(jìn)行綜合評價。結(jié)果以Rv2031c、38kDa和CFP10-ESAT6為包被抗原的ELISA法檢測相應(yīng)抗體的靈敏度分別為41.0%,51.3%和46.2%,特異度分別為90.0%、88.9%和79.1%,曲線下面積分別為0.640、0.761和0.690,以3種蛋白混合物為包被抗原的3項指標(biāo)分別為66.7%、77.8%和0.746。結(jié)論 Rv2031c、38kDa以及融合蛋白CFP10-ESAT6 3種抗原混合物的診斷效能較好,具有一定的臨床應(yīng)用價值。
[Abstract]:Objective to evaluate the efficiency of detecting the mixture of Rv2031cP38kDa and fusion protein CFP10-ESAT6, and to screen the best antigen and analyze its application value. Methods the target gene was amplified from the genome of Mycobacterium tuberculosis standard strain H37Rv, and the recombinant plasmid was constructed by pET-30a pET-32a vector. The recombinant protein was induced and expressed in host E.coli BL21 (DE3) cells. The recombinant protein was purified by His tag affinity nickel column. ELISA was used to detect 129 sera (39 sera from tuberculosis patients and 90 sera from healthy people). The optimal critical value (Cut-off value) of different antigens and mixed antigens (Cut-off value) was calculated by using the method of operating characteristic curve. The sensitivity, specificity and area under the curve were obtained. The diagnostic efficacy and application value were evaluated synthetically. Results the sensitivities of Rv2031cn38kDa and CFP10-ESAT6 for the detection of the corresponding antibodies were 41. 0% and 46. 2%, the specificity was 90. 088. 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 coated antigens were 67. 8% and 0. 746, respectively. Conclusion Rv2031 ct 38kDa and fusion protein CFP10-ESAT6 have good diagnostic efficacy and have certain clinical application value.
【作者單位】： 北京市朝陽區(qū)疾病預(yù)防控制中心;傳染病預(yù)防控制國家重點實驗室中國疾病預(yù)防控制中心傳染病預(yù)防控制所/傳染病預(yù)防控制國家重點實驗室;
【基金】：國家"艾滋病和病毒性肝炎等重大傳染病防治科技重大專項"課題(No.2013ZX10003006)資助
【分類號】：R52

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