五个结核分枝杆菌重组蛋白及其组合物的细胞免疫评价

Cellular immunity evaluation of five mycobacterium tuberculosis recombinant proteins and their compositions

目的:对5个结核分枝杆菌重组蛋白及其组合物的细胞免疫进行评价。方法:88份新鲜肝素抗凝人群外周静脉血液,其中42份来自北京市昌平区结核病防治所就诊的结核病患者,46份健康志愿者由中国疾病预防控制中心传染病所提供,均为接种过卡介苗(BCG)且没有结核暴露史和任何临床症状体征的健康人。以结核分枝杆菌标准菌株H37Rv DNA为模板,PCR扩增选取的5个重组蛋白Rv3874、Rv3875、Rv2031c、Rv1411c和Rv3418c的完整编码基因;通过基因重组和蛋白纯化技术,分别克隆、表达和纯化5个重组蛋白作为刺激物,采用效应T细胞酶联免疫斑点试验(ELISPOT)对人群进行细胞免疫检测。结果:成功克隆、表达和纯化了Rv3874、Rv3875、Rv2031c、Rv1411c和Rv3418c重组蛋白;灵敏度分别为50.00%、71.43%、69.04%、73.81%和76.19%;特异度分别为86.96%、76.09%、71.74%、39.13%和36.96%。阳性预测值、阴性预测值、曲线下面积及约登指数分别为52.46%~77.78%、62.96%~74.47%、0.511~0.754和0.129~0.475。除Rv1411c和Rv3418c外,Rv3874、Rv3875和Rv2031c在结核病患者中检测到的斑点形成细胞数均高于健康志愿者,差异均具有统计学意义(P值均<0.001)。在5个重组蛋白所构成的26种组合物中,灵敏度为80.95%~95.24%,特异度为68.89%~24.44%。随着组合物中重组蛋白个数的增加其灵敏度逐渐增加,但特异度随之下降。结论:结核分枝杆菌重组蛋白Rv3874、Rv3875和Rv2031c刺激T细胞产生免疫应答的能力较强,具有一定的抗原性。而Rv1411c和Rv3418c单独用作诊断抗原的效能并不理想,与多组分抗原联合构成的组合物具有一定应用价值。

Objective The cellular immunity of 5 Mycobacterium tuberculosis recombinant proteins and their compositions was evaluated.Method A total of 88 fresh venous blood from peripheral heparin anticoagulant population,42 of which were from tuberculosis patients treated by The Tuberculosis Prevention and Treatment Center of Changping District,Beijing,and 46 of healthy volunteers were provided by the Infection Diseases of Chinese Center for Disease Control and Prevention.Healthy volunteers without a history of tuberculosis exposure and any clinical signs and symptoms.Using the Mycobacterium tuberculosis standard strain H37Rv DNA as a template,complete genes of the selected 5 recombinant proteins Rv3874,Rv3875,Rv2031c,Rv1411c and Rv3418c by PCR amplified;5 recombinant proteins were cloned,expressed and purified as stimulants by genetic recombination and protein purification techniques,and the effector T cell enzyme-linked immunospot assay(ELISPOT)was used to detect cellular immunity in the population.Results The recombinant proteins Rv3874,Rv3875,Rv2031c,Rv1411c and Rv3418c were successfully cloned,expressed and purified;And the sensitivities were 50.00%,71.43%,69.04%,73.81%and 76.19%,and the specificities were 86.96%,76.09%,71.74%,39.13%and 36.96%.In addition,the positive predictive value,negative predictive value,area under the curve and Youden index were 52.46%to 77.78%,62.96%to 74.47%,0.511 to 0.754 and 0.129 to 0.475,respectively.Except for Rv1411c and Rv3418c,the number of spot-forming cell(SFC)detected by Rv3874,Rv3875 and Rv2031c in tuberculosis patients was higher than healthy volunteers,and the differences were statistically significant(P<0.001).Among the 26 compositions composed of 5 recombinant proteins,the sensitivity was 80.95%to 95.24%,and the specificity was 68.89%to 24.44%.As the number of recombinant proteins in the composition increases,the sensitivity gradually increased,but the specificity decreased.Conclusion The recombinant proteins of Mycobacterium tuberculosis Rv3874,Rv3875 and Rv2031c have strong ability to stimulate T cells to produce immune response,and have certain antigenicity.The efficacy of Rv1411c and Rv3418c alone as diagnostic antigens is not ideal,and the composition composed of multi-component antigens has certain application value.This article provides experimental evidence for the immune diagnosis of tuberculosis and the preparation of new anti-tuberculosis vaccines.