结核分枝杆菌rHpsX-MPT64融合蛋白的制备与临床应用

目的构建结核分枝杆菌融合基因hpsX-mpt64原核表达系统,建立基于rHpsX-MPT64为包被抗原酶联免疫吸附测定法(Enzyme-linked immunosorbent assay,ELISA)并对其用于结核患者血清学诊断的敏感性和特异性进行评价。方法PCR扩增并克隆结核分枝杆菌hpsX和mpt64基因,以连接引物PCR构建hpsX-mpt64人工融合基因,建立上述目的基因原核表达系统。采用SDS-PAGE检测目的重组蛋白rHpsX、rMPT64和rHpsX-MPT64表达情况,Ni-NTA亲和层析法提纯上述目的重组蛋白,Western Blot检测其免疫性。分别以rHpsX、rMPT64和rHpsX-MPT64为包被抗原,建立相应ELISA检测150例健康人群、73例非结核肺部疾病患者和277例活动性肺结核患者血清标本中抗体。结果克隆的hpsX和mpt64及构建的hpsX-mpt64融合基因与GenBank中相关序列相似性为100%。rHpsX、rMPT64和rHpsX-MPT64表达量分别为细菌总蛋白的20.3%、28.5%和26.5%,其提纯物电泳后均显示单一条带。基于rHpsX、rMPT64和rHpsX-MPT64为包被抗原的ELISA对涂阳患者血清抗体检出率明显高于涂阴患者(P<0.01),检测活动性结核患者血清抗体的敏感性分别为59.2%、61.4%和74.4%,特异性分别为90.6%、83.9%和91.0%,rHpsX-MPT64为包被抗原敏感性明显高于rHpsX和rMPT64抗原(P<0.01)。结论基于rHpsX-MPT64为包被抗原的ELISA有较高的敏感性和特异性,重组融合蛋白为抗原有助于进一步提高检测结核病血清学诊断的敏感性。

Construction and Expression of Prokaryotic Expression Vector of MPT-64 Gene

[Objective]Protective antigen gene MPT-64 was cloned from genomic DNA of Mycobacterium tuberculosis and transferred into prokaryotic competent cells for expression to obtain MPT-64 fusion protein.[Method]Based on the GenBank,primers were designed for amplification of MPT-64 gene,and the recombinant plasmid pET-32a-MPT-64 was constructed.The recombinant plasmid was expressed in prokaryotic expression vector to obtain fusion protein.[Result]Protective antigen gene MPT-64 was successfully cloned.The recombinant plasmid pET-32a-MPT-64 was obtained.MPT-64 fusion protein was successfully expressed.[Conclusion]This study laid solid foundation for the prevention,diagnosis,treatment of tuberculosis and the development of tuberculosis vaccines.