SARS冠状病毒核衣壳蛋白的克隆表达及其临床应用研究

Molecular cloning and expression of the severe acute respiratory syndrome-associated coronavirus nucleocapsid protein and its clinical application

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 。

Objective To clone and express nucleocapsid (N) protein of the severe acute respiratory syndrome (SARS)-associated coronavirus, and to evaluate its antigenicity and application value in the development of serological diagnostic test for SARS. Methods SARS-associated coronavirus N protein gene was amplified from its genomic RNA by reverse transcript nested polymerase chain reaction (RT-nested-PCR) and cloned into pBAD/Thio-TOPO prokaryotic expression vector. The recombinant N fusion protein was expressed and purified,and its antigenicity and specificity was analyzed by Western Blot,to establish the recombinant N protein-based ELISA for detection of IgG antibodies to SARS-associated coronavirus,and SARS-associated coronavirus lysates-based ELISA was compared parallelly. Results The recombinant expression vector produced high level of the N fusion protein after induction,and that protein was purified successfully by affinity chromatography and displayed higher antigenicity and specificity as compared with whole virus lysates. Conclusion The recombinant SARS-associated coronavirus N protein possessed better antigenicity and specificity and could be employed to establish a new,sensitive,and specific ELISA for SARS diagnosis.