【摘要】 目的 克隆并表达结核分枝杆菌（MTB）重组融合蛋白磷酸盐转运系统蛋白1-热休克蛋白16.3(PstS1-Hsp16.3)，建立基于重组融合蛋白PstS1-Hsp16.3的纳米金免疫传感器检测结核病患者血清抗体。方法 重叠延伸PCR扩增融合基因pstS1-hsp16.3，构建重组表达质粒pET-28a(+)-pstS1-hsp16.3，诱导表达并纯化融合蛋白PstS1-Hsp16.3，免疫印迹分析其反应原性。将制备的金纳米棒经化学修饰后，与目的蛋白连接，构建纳米金免疫传感器检测结核病患者血清抗体（收集2018年7月-2019年1月湖南省胸科医院检验科50例结核病患者血清，以及湖南省人民医院检验科50名健康者血清及42例非结核肺病患者血清）。结果 重叠延伸PCR扩增获得约1 570 bp的融合基因片段，pMD18-T-pstS1-hsp16.3阳性克隆的测序结果与GenBank报道的MTB H37Rv株pstS1和hsp16.3基因序列完全一致。重组表达质粒pET-28a(+)-pstS1-hsp16.3的双酶切及菌落PCR均获得约1 570 bp的基因片段。构建的pET-28a(+)-pstS1-hsp16.3并经表达纯化获得约60×10~3 Mr的目的蛋白。免疫印迹结果显示，纯化的PstS1-Hsp16.3可与结核病患者血清反应，与健康人群血清不反应，表明其拥有较好的反应原性。建立了基于重组融合蛋白PstS1-Hsp16.3的纳米金免疫传感器，与结核病患者混合血清、健康人群混合血清、非结核肺病患者混合血清反应后，纵向等离子体吸收峰的波长位置由788 nm分别变为801、790、790 nm，红移了13、2、2 nm，表明存在抗原抗体特异性反应。将该传感器对结核病患者血清、健康人群血清及非结核肺病患者血清进行检测，接受者操作特性曲线显示其检测的灵敏度和特异度分别为82%和80%。结论 基于融合蛋白PstS1-Hsp16.3的纳米金免疫传感器可辅助诊断结核病。更多还原

【Abstract】 Objective To express the recombinant fusion protein phosphate specific transport system 1-heat shock protein 16.3(PstS1-Hsp16.3) of Mycobacterium tuberculosis(MTB) for the detection of the serum antibody of patients with tuberculosis. Methods The fusion gene pstS1-hsp16.3 was amplified by overlapping extension PCR and cloned into the prokaryotic expression vector pET28a(+) to construct the recombinant expression plasmid pET28a(+)-pstS1-hsp16.3. The fusion protein PstS1-Hsp16.3 was purified by metal chelate chromatography and its reactogenicity was analyzed by immunoblotting. The gold nanorods prepared by crystal growth method were chemically modified and attached to the fusion protein PstS1-Hsp16.3 so as to construct a nanogold immunosensor for detection of serum antibodies(Serum from 50 tuberculosis patients was collected from the laboratory of the Hunan Chest Hospital, and serum from 50 healthy people and 42 non-tuberculous pulmonary disease patients was collected from the laboratory of the Hunan Provincial People’s Hospital from July 2018 to January 2019). Results The fusion gene fragment of about 1 570 bp was amplified by overlapping extension PCR, and the sequencing results of the positive clones of pMD8-T-pstS1-hsp16.3 were completely consistent with the pstS1 and hsp16.3 gene sequences of MTB H37 Rv strains reported in GenBank. The double enzyme digestion and colony PCR of the recombinant expression plasmid pET-28a(+)-pstS-hsp16.3 both obtained a DNA fragment of about 1 570 bp. The constructed pET-28a(+)-pstS1-hsp16.3 was expressed and purified to obtain the target protein with an approximate molecular weight of 60×103 Mr, and the results of immunoblot showed that the purified PstS1-Hsp16.3 could react with the serum antibodies of tuberculosis patients, but not with the serum antibodies of healthy people, indicating that it had a better reactogenicity. The nanogold immunosensor based on the recombinant fusion protein PstS1-Hsp16.3 reacted with the mixed sera of tuberculosis patients, healthy people and non-tuberculosis pulmonary disease patients, respectively, the wavelength positions of longitudinal plasma absorption peak changed from 788 nm to 801, 790 and 790 nm respectively, and the red shifts were 13, 2 and 2 nm, indicating the existence of antigen-antibody specific reaction. The sensor was used to detect the serum antibodies of tuberculosis patients, healthy people and non-tuberculosis pulmonary disease patients; the receiver operating characteristic curve showed that the sensitivity and specificity were 82% and 80%, respectively. Conclusion The nanogold immunosensor based on the fusion protein PstS1-Hsp16.3 could be used for auxiliary diagnosis of tuberculosis.更多还原