Cloning and Expression of Immunoreactive Antigens from Mycobacterium tuberculosis

Four immunoreactive proteins, B.4, B.6, B.10, and B.M, with molecular weights ranging from 16,000 to 58,000, were observed from immunoblots of Mycobacterium tuberculosis total lysates screened with sera from individuals with active tuberculosis. These proteins were identified from microsequence analyses, and genes of proteins with the highest homology were PCR amplified and cloned into the pQE30 vector for expression studies. In addition, a 37.5-kDa protein, designated C17, was identified from a phage expression library of M. tuberculosis genomic DNA. Preliminary immunoblot assays indicated that these five resultant recombinant proteins could detect antibodies in individuals with active pulmonary and extrapulmonary tuberculosis. The overall ranges of sensitivities, specificities, positive predictive values, and negative predictive values for the recombinant antigens were 20 to 58, 88 to 100, 69 to 100, and 56 to 71%, respectively. The B.6 antigen showed preferential reactivity to antibodies in pulmonary compared to nonpulmonary tuberculosis serum specimens. All of these recombinant antigens demonstrated potential for serodiagnosis of tuberculosis.

采用活动性结核病患者血清对结核分枝杆菌（Mycobacterium tuberculosis）全裂解物进行免疫印迹（immunoblot）筛查，共检出4种免疫反应性蛋白，分别为B.4、B.6、B.10及B.M，其分子量介于16000至58000之间。上述蛋白通过微序列分析（microsequence analysis）完成鉴定，随后对同源性最高的蛋白编码基因进行聚合酶链式反应（PCR）扩增，并克隆至pQE30载体以开展表达研究。此外，从结核分枝杆菌基因组DNA的噬菌体表达文库中鉴定出一种命名为C17的37.5 kDa蛋白。初步免疫印迹实验结果显示，这5种重组蛋白可检测活动性肺结核及肺外结核病患者体内的抗体。该批重组抗原的敏感性、特异性、阳性预测值及阴性预测值的总体区间分别为20%~58%、88%~100%、69%~100%及56%~71%。相较于肺外结核血清标本，B.6抗原对肺结核患者体内抗体的反应性更具优势。上述所有重组抗原均具备结核病血清学诊断的应用潜力。