重组结核分枝杆菌Hsp16.3的纳米金免疫传感器检测Hsp16.3抗体

克隆和表达结核分枝杆菌热休克蛋白16.3(Hsp16.3),建立纳米金免疫传感器检测结核病患者血清Hsp16.3抗体。PCR扩增hsp16.3基因,构建重组表达质粒pQE30-hsp16.3,表达和纯化Hsp16.3,Western blot分析其反应原性;晶种生长法制备金纳米棒并连接Hsp16.3,建立纳米金免疫传感器检测血清Hsp16.3抗体,接受者操作特性(ROC)曲线评价其灵敏度和特异性。成功构建重组表达质粒pQE30-hsp16.3,纯化Hsp16.3具有良好的反应原性;Hsp16.3的纳米金免疫传感器分别检测50例结核病患者、42例非结核肺病患者和50例体检健康者血清,红移值大于3.5的例数分别为42、7、1例;ROC曲线显示,曲线下面积0.888(P0.01),界值3.5,灵敏度82%,特异性98%。结果表明,Hsp16.3的纳米金免疫传感器可用于检测结核病血清Hsp16.3抗体。

Detection of Antibodies to Hsp16.3 in Serums of Tuberculosis by Gold Nanorod Immunosensor with Recombinant Hsp16.3 of Mycobacterium tuberculosis

Cloning and expression of heat shock protein 16.3 (Hsp16.3) of Mycobacterium tuberculosis and detection of antibodies to Hsp16.3 by gold nanorod immunosensor with recombinant Hsp16.3 were carried out. The gene fragment of hsp16.3 amplified by PCR was conjugated with prokaryotic expression vector pQE30 to construct a recombinant expression plasmid of pQE30-hsp16.3. Hsp16.3 was expressed and purified from the recombinant E. coli JM109 containing the plasmid of pQE30-hsp16.3. The reactivity of Hsp16.3 was analyzed with Western blot. Gold nanorods were prepared by the seeded growth method and connected with the Hsp16.3 to construct gold nanorod immunosensor for the detection of antibodies to Hsp16.3 in the serums of tuberculosis and controls; receiver operating characteristic (ROC) curve was used to evaluate its sensitivity and specificity in the serums of tuberculosis. The results showed that the recombinant expression plasmid of pQE30-hsp16.3 was successfully constructed, and the purified Hsp16.3 was shown with good reactivity. The immunosensor was respectively used to detect the serums of 50 tuberculosis patients, 42 nontuberculous pulmonary disease patients, and 50 healthy subjects and the number whose red shift value was more than 3.5, 42, 7 and 1 respectively. ROC curve showed that the area under the curve was 0.888 (P<0.01); cut-off value 3.5, sensitivity 82% and specificity 98%. Therefore, gold nanorod immunosensor with recombinant Hsp16.3 can be used to detect antibodies Hsp16.3 in serums of tuberculosis.