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目的 克隆、原核表达严重急性呼吸综合征冠状病毒 (SARS-CoV)核衣壳 (N)蛋白 ,分析、评价其抗原性和在SARS血清学诊断中的应用价值。方法 采用逆转录巢式聚合酶链反应 (RT-nested PCR)扩增SARS CoV的N蛋白基因 ,克隆入pBAD-Thio TOPO原核表达载体 ,表达、纯化重组融合N蛋白 ,WesternBlot分析其抗原性和特异性 ,建立以重组N蛋白为抗原的酶联免疫吸附测定(ELISA)法 ,并与以全病毒裂解液为抗原的ELISA法进行比较。结果 重组表达载体经诱导产生了高水平的重组融合N蛋白 ,融合蛋白经亲和纯化后 ,具备了较高的纯度和抗原反应性 ,以重组蛋白为抗原的ELISA法在特异性和敏感性方面优于以全病毒裂解液为抗原者。结论 重组SARS-CoV的N蛋白具有良好的抗原性和特异性 ,可作为新一代SARS-CoV抗体检测试剂盒的备选抗原
Objective To clone and express the nucleocapsid protein of severe acute respiratory syndrome (SARS-CoV) nucleocapsid (N) in prokaryotic cells, analyze and evaluate its antigenicity and its value in serological diagnosis of SARS. Methods The N protein gene of SARS CoV was amplified by RT-nested PCR and cloned into prokaryotic expression vector pBAD-Thio TOPO. The recombinant fusion N protein was expressed and purified. The antigenicity and specificity To establish an enzyme-linked immunosorbent assay (ELISA) method using recombinant N protein as an antigen, and compare the ELISA method with the whole virus lysate as an antigen. Results The recombinant fusion protein was induced to produce a high level of recombinant fusion protein N, and the fusion protein was purified by affinity chromatography with high purity and antigenic reactivity. The ELISA method using recombinant protein as antigen was highly sensitive and specific Better than whole virus lysate as antigen. Conclusion The N protein of recombinant SARS-CoV has good antigenicity and specificity and can be used as a candidate antigen of the new generation SARS-CoV antibody detection kit