中国首次分离的辛德毕斯病毒XJ-160病毒株感染性全基因组cDNA克隆的构建与分析

Construction and Infection Analysis of the Full-length cDNA Clone of XJ-160 Virus,the First Sindbis Virus Isolated in China

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SP6 RNA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DH5a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(8 453位核苷酸由C变为T)产生Xba Ⅰ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.

We reported the full-length infectious cDNA clone of XJ-160 strain of Sindbis virus firstly isolated in China. The full-length cDNA was constructed from reverse transcription-PCR products of viral RNA and cloned into plasmid derived from pBR322 under the control of a SP6 promoter and followed by a polyA tail behind 3 terminus of genomic cDNA. It was stably amplified in Escherichia coli DH5. RNA transcribed from the full-length cDNA clone was highly infectious, after transfection into BHK-21 cell, resulting in generation of recovered progeny virus with titre of 10~7-10~8PFU/ml.A silent nucleotide change was introduced into the cDNA clone to create a Xba I site (C to T at 8453nt) that is used as a genetic marker of this infectious clone. This genetic marker was retained in the genome of recovered progeny virus. The recovered virus was indistinguishable from the parental virus XJ-160 in the aspects of cytopathogenic effect, plaque morphology on BHK-21cells, viral antigenicity, in vitro growth characteristics in BHK-21 cells and the virulence in suckling mice. The stable infectious cDNA clone of XJ-160 strain, as an effective reverse genetics experimental system, will provide a valuable molecular biological tool to study the pathogenesis and replication of Sindbis virus and to develop the vector system of Alphavirus.