SARS_CoV中国株基因组全长cDNA的构建及其恢复病毒的生物学性质

Construction of genomic full_length cDNA of SARS coronavirus BJ01 strain and identification of its biological characteristics

严重急性呼吸综合征是SARS-CoV引起的一种重要新发传染病,其致病机制的研究对于防治该病十分必要。为了利用反向遗传学技术研究SARS-CoV的致病机制,将覆盖SARS-CoVBJ01株基因组全长的7个cDNA片段纯化后进行体外连接,构建基因组全长cDNA分子,以其为模板,使用T7RNA聚合酶系统在体外进行转录,获得病毒RNA。用电穿孔转染法将转录体RNA导入VeroE6细胞,可观察到典型的SARS-CoV致细胞病变作用。对收获的恢复病毒采用RT-PCR方法进行鉴定,结果表明获得的恢复病毒与SARS-CoVBJ01株原病毒序列一致。以针对SARS-CoV的抗体对感染细胞作间接免疫荧光反应,证明获得了具有特异感染性的恢复病毒。同时用细胞病变法和空斑试验测定了恢复病毒及其亲本毒株的病毒滴度,结果表明二者在致病性上没有明显差异,恢复病毒具有与原型株相似的生物学特性。SARS-CoVBJ01株基因组全长cDNA的成功构建及对恢复病毒生物学性质的研究将为进一步探索SARS-CoV致病的分子机制及研制新型疫苗奠定良好的基础。

Severe acute respiratory syndrome （SARS） is an important emerging infectious disease which caused by SARS coronavirus （SARS-CoV）, and the study of its pathogenesis is needed for the treatment and prevention of this disease. To study the pathogenesis of SARS-CoV using reverse genetics technology, by in vitro ligation using 7 contiguous cDNAs that span the entire genome of the SARS-CoV BJ01 strain, a genomic full-length cDNA was assembled, then using T7 RiboMAXTM Large Scale RNA Production Systems with the genomic full-length cDNA as template, the RNA transcript was attained. The typical SARS-CoV-resulted cell pathogenic effects were observed when RNA transcript was electroporated into Vero E6 cells. The results of RT-PCR and sequencing of the rescued virus showed that it originated from transcript which derived from the full-length cDNA construct. Rescued virus-infected ceils were detected by indirect fluorescent antibody staining demonstrated that it can specifically reaction with SARS-CoV. By CPE method and plaque assay, the titers of the rescued virus and wild-type virus were assessed, which demonstrated there are no significant difference between the viruses and they have similar biological characteristics. Construction of the genomic full-length cDNA of SARS-CoV BJ01 stain successfully and study of the biological characteristics of the rescued virus will provide a useful tool serving for the discovery of molecular pathogenesis and development of candidate vaccines against SARS-CoV.