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

作　　者：杨益良[1] 梁国栋[1] 付士红[1] 何海怀[1] 李晓宇[1] 邓娟[1] 苏乃伦[1] 王力华[1] 侯云德[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所病毒基因工程国家重点实验室,北京100052

出　　处：《病毒学报》2005年第3期173-180,共8页Chinese Journal of Virology

基　　金：国家自然科学基金资助(No.39970037)

摘　　要：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.

关 键 词：感染性全基因组cDNA克隆 恢复病毒 辛德毕斯病毒 XJ-160病毒株 中国 分离 

分 类 号：R373.3[医药卫生—病原生物学]