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从一例输入性传染性非典型性肺炎病人血清中提取病毒RNA,通过RT-PCR方法扩增出SARS病毒核蛋白基因片段,克隆入质粒载体pUCm-T后,进行核苷酸序列的测定及分析,与已公布的SARS病毒基因序列进行比较,证实为SARS冠状病毒核蛋白基因。为了解该病毒核蛋白的抗原特性,将核蛋白基因插入表达载体,构建重组质粒pET28a-SN,转导大肠杆菌BL21(DE3)后,加IPTG诱导表达。产物经SDS-PAGE电泳分析,表达出相对分子量约为50kDa的蛋白,占整个菌体的45％左右。Western-blot分析表明,表达产物仅与SARS阳性病人血清起反应,而与正常血清不起反应。间接ELISA免疫检测,抗原滴度达1:12500。表明表达的核蛋白为SARS特异性抗原,这为SARS病毒的诊断试剂的研制提供了方便而安全的抗原来源。
The virus RNA was extracted from the serum of a patient with SARS and amplified by RT-PCR from the nucleocapsid gene of SARS virus. After cloned into the plasmid vector pUCm-T, the nucleotide sequence was determined and analyzed , Compared with the published SARS virus gene sequence, confirmed as SARS coronavirus nucleoprotein gene. To understand the antigenicity of the viral nucleoprotein, the nucleoprotein gene was inserted into the expression vector to construct the recombinant plasmid pET28a-SN. The recombinant plasmid pET28a-SN was induced by IPTG after it was transformed into E. coli BL21 (DE3). The product was analyzed by SDS-PAGE electrophoresis, and expressed a relative molecular weight of about 50kDa protein, accounting for about 45% of the entire cell body. Western-blot analysis showed that the products of expression only reacted with the serum of SARS-positive patients, but not with the normal serum. Indirect ELISA immunoassay, antigen titer of 1: 12500. The result showed that the expressed nucleoprotein was SARS-specific antigen, which provided a convenient and safe source of antigen for the development of SARS virus diagnostic reagent.