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。

Preparation and clinical application of recombinant fusion protein rHpsX-MPT64 of Mycobacterium tuberculosis

In this study, we constructed the prokaryotic expression systems of hpsX and mpt64 genes and to establish enzyme-linked immunosorbent assay （ELISA） based on rHpsX-MPT64 fusion antigen of M. tuberculosis （TB） in order to eval uate the sensitivity and specificity of the ELISA for detection of serological diagnosis of TB patients. The hpsX and rapt64 genes were amplified and cloned. A PCR with linking primers was used to construct artificial fusion gene hpsX-mpt64. The prokaryotic expression systems of the genes were then constructed. SDS-PAGE was used to measure the expression of the tar- get recombinant proteins rHpsX, rMPT64 and rHpsX-MPT64. Ni-NTA affinity chromatography was applied to extract the three recombinant proteins. By using rHpsX, MPT64 and rHpsX-MPT64 as the coated antigens, ELISA was used to detect serum samples from 150 healthy individuals, 73 patients with other pulmonary diseases, and 277 pulmonary tuberculosis cases. The results of sequencing similarities of the cloned hpsX and mpt64 genes and the constructed hpsX-mpt64 fusion gene were 100% compared with the corresponding sequences in GenBank. The expression outputs of rHpsX, rMPT64 and rHpsX- MPT64 were approximately 20.3%, 28.5% and 26.5% of the total bacterial proteins, respectively. Each of the three purified recombinant proteins showed a single fragment in gel after electrophoresis. The positive rates of ELISA using rHpsX, rMPT64 or rHpsX-MPT64 as the antigen for the serum samples in the bacterium-positive TB patients were stronger than that in the bac- terium-negative （P 〈 0. 01 ）. The sensitivity of ELISA using rHpsX, rMPT64 or rHpsX-MPT64 as the antigens for detecting serum antibodies of TB patients were 59. 2%, 61. 4% and 74.4%, respectively. The specificity of ELISA using rHpsX,rMPT64 and rHpsX-MPT64 as the antigens for detecting serum antibodies of TB patients were 90.6%, 83.9% and 91.0%, respectively. The sensitivity of ELISA using rHpsX-MPT64 as the antigen was higher than that of ELISA using rHpsX and rMPT64 as the antigens. It＇s concluded that rHpsX-MPT64-ELISA has higher sensitivity and specificity in serodiagnosis of tu- berculosis, and the fusion rHpsX-MPT64 antigen might improve the sensitivity of serological diagnosis of TB.