SARS病毒核蛋白基因的克隆及其表达研究

Cloning and Expression of Nucleocapsid Protein Gene of SARS Associated Coronavirus

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。

Nucleocapsid gene of SARS-associated coronavirus(SARS-CoV) was obtained by reverse transcription and polymerase chain reaction from a pateint suffered from severe acute respiratory syndrome (SARS) from Beijing, subsequently cloned into pUCm-T vector. The sequence of positive recombinants was determined by the method of dideoxy chain termination, which revealed that the nucleocapsid gene segment is 1269 nucleotide in length with an open reading frame encoding a protein of 422 amino acids. Nucleocapsid gene was subcloned into the prokaryotic vector pET28a. The recombinant plasmid pET28a-SN was transformed into host cell BL21 (DE3). After inducing by IPTG, about 50kD protein was expressed and the expression level was about 45%. Western-blot analysis demonstrated that the recombinant proteins reacted with SARS positive sera but not with normal sera tested. SARS nucleocapsid protein expressed by the E.coli system offer a safe source of specific antigen for diagnostic purposes.