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报告了中国首次分离的辛德毕斯病毒XJ-160株的感染性全基因组cDNA克隆的构建与鉴定。利用RT-PCR方法获得覆盖病毒全长基因组的cDNA片段,以低拷贝质粒pBR322作为骨架,将基因组cDNA置于SP6RNA聚合酶启动子之后,基因组3′末端带有35个连续的A,通过DNA重组技术组装成病毒基因组全长cDNA克隆。该克隆可在大肠杆菌DH5α中稳定扩增。经体外转录,RNA转录体转染BHK-21细胞,细胞发生病变,恢复病毒滴度达到107~108PFU/ml。全基因组cDNA克隆构建过程中引入的沉默突变(8453位核苷酸由C变为T)产生XbaI酶切位点作为遗传标记,在子代恢复病毒的基因组中稳定存在。从细胞病变的特征、BHK-21细胞的空斑形态、病毒的抗原性、病毒在细胞中的生长动力学特征以及对乳鼠的致病性等方面比较,恢复病毒和亲本病毒XJ-160没有显著区别,提示获得了具有感染性的XJ-160病毒全长cDNA克隆。该病毒感染性全基因组cDNA克隆可以作为反向遗传学系统,为进一步研究病毒复制和致病机制,以及开发相应的载体表达系统提供分子生物学工具。
The construction and identification of the infectious genome-wide cDNA clone of Sindbis virus XJ-160, which was first isolated in China, was reported. The cDNA fragment covering the full-length genome of the virus was obtained by RT-PCR. After the low-copy plasmid pBR322 was used as the backbone, the genomic cDNA was placed in the promoter of SP6 RNA polymerase with 35 contiguous A at the 3 ’end of the genome, The technology is assembled into a full-length cDNA clone of the viral genome. This clone is stably amplified in E. coli DH5α. After in vitro transcription, RNA transcripts were transfected into BHK-21 cells and the cells developed lesions, and the virus titer was restored to 107-108 PFU / ml. The silent mutation (8453 nucleotides from C to T) introduced during the construction of whole genome cDNA cloning generated the XbaI restriction site as a genetic marker, which was stable in the genome of offspring recovering virus. The virus and the parental virus XJ-160 were recovered from the characteristics of cytopathic effects, the plaque morphology of BHK-21 cells, the antigenicity of the virus, the growth kinetic characteristics of the virus in the cells, and the pathogenicity of the suckling mice Significant difference, suggesting that the infectious XJ-160 virus full-length cDNA clone was obtained. The infectious genome-wide cDNA clone can be used as a reverse genetics system to provide molecular biology tools for further research on viral replication and pathogenesis and to develop corresponding vector expression systems.