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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 is an important emerging infectious disease caused by SARS-CoV. The study of its pathogenesis is very necessary to prevent and treat this disease. In order to study the pathogenesis of SARS-CoV by using reverse genetics technique, seven cDNA fragments covering the entire genome of SARS-CoVBJ01 strain were purified and ligated in vitro to construct a full-length genomic cDNA molecule, which was used as a template and T7RNA The polymerase system transcribes in vitro to obtain viral RNA. Transfection of transcriptome RNA into VeroE6 cells by electroporation transfection allowed the observation of a typical SARS-CoV cytopathic effect. The harvested recovered virus was identified by RT-PCR and the results showed that the obtained recovered virus was consistent with the original SARS-CoVBJ01 strain. Indirect immunofluorescence reaction of infected cells against SARS-CoV antibody proved that a specifically infectious retrovirus was obtained. The virus titer of the recovered virus and its parent strain was determined by cytopathic assay and plaque assay. The results showed that there was no significant difference in the pathogenicity of the recovered virus and the recovered virus had similar biological characteristics to the original strain. The successful construction of the full-length cDNA of SARS-CoVBJ01 strain and the study of the biological properties of the recovered virus will lay a good foundation for further exploration of the molecular mechanism of SARS-CoV pathogenesis and development of new vaccines.