nsP2-726Pro定点诱变对辛德毕斯病毒XJ-160复制子载体特性的影响  

Effects of the Site-directed Mutagenesis at nsP2-726Pro on Replicon Vector Derived from XJ-160 Virus

作　　者：唐丽[1,2] 朱武洋[2] 付士红[2] 何英[2] 王志玉[1] 梁国栋[2] 

机构地区：[1]山东大学公共卫生学院,济南250012 [2]中国疾病预防控制中心病毒病预防控制所传染病预防控制国家重点实验室,北京100052

出　　处：《病毒学报》2010年第2期121-127,共7页Chinese Journal of Virology

基　　金："艾滋病和病毒性肝炎等重大传染病防治"科技重大专项(2008ZX10004-001A;2009ZX10004-705);国家自然基金(30170046;30970160)

摘　　要：为观察nsP2-726Pro定点诱变对辛德毕斯病毒(Sindbis virus,SINV)XJ-160复制子载体特性的影响,以XJ-160病毒复制子载体pBRepXJ为分子基础,利用定点诱变方法分别构建突变载体pBRep-726L、pBRep-726S、pBRep-726V和pBRep-726A。将新霉素抗性基因(Neomycinr,Neor)克隆到pBRepXJ和各突变载体中,通过细胞培养方法观察nsP2-726Pro定点诱变对载体致细胞病变作用(Cytopathic effect,CPE)的影响;并将增强型绿色荧光蛋白(Enhanced green-fluorescent protein,EGFP)和海肾萤光素酶(Renilla Luciferase,R.luc)报告基因分别插入到各载体中,定性与定量检测定点诱变对复制子载体自主复制能力的影响。实验结果表明,突变载体pBRep-726V和pBRep-726A在BHK-21细胞中的自主复制能力高于pBRepXJ,所诱发的细胞病变进程更快。替换突变nsP2-726Pro→Leu的引入使载体在保持包装能力的同时,完全丧失在细胞上引起CPE的能力。而pBRep-726S则具有中间表型。提示nsP2-726Pro定点诱变在影响XJ-160复制子载体自主复制能力的同时,也改变了CPE的进程。这将为研究辛德毕斯病毒基因组结构与功能的关系及构建非细胞病变的甲病毒载体奠定基础。To investigate the effects of site-directed mutagenesis at nsP2-726Pro on the characteristics of replicon vector derived from XJ-160 virus,a Sindbis virus （SINV） isolated in China. The mutant vector pBRep-726L,pBRep-726S,pBRep-726V or pBRep-726A was constructed by introducing nsP2-726Pro→Leu,nsP2-726Pro→Ser,nsP2-726Pro→Val or nsP2-726Pro→Ala into XJ-160 viral replicon vector pBRepXJ respectively. To quantitatively and qualitatively determine the site-directed mutagenesis on the replicon,the recombinant plasmids expressing Neomycinr （Neor）,enhanced green fluorescent protein （EGFP） or Renilla luciferase （R.luc） were constructed by cloning report genes into pBRepXJ or mutant XJ-160 vector respectively. And in vitro-synthesized RNA from expression vectors were electroporated into BHK-21 cells. Compared with the wild-type replicon,the mutation nsP2-726Pro→Val or nsP2-726Pro→Ala accelerated the processing of CPE on BHK-21 cells and simultaneously enhanced its self-replicating capacity. The mutant vector pBRep-726L with Leu substitution exhibited similar packaging capacity to that of pBRepXJ. In contrast,pBRep-726S exhibited a medium phenotype,including the process of CPE and the activity of R.luc expression in BHK-21 cells. The site-directed mutagenesis at nsP2-726Pro not only regulates directly XJ-160 virus vector-host cell interactions,but also plays an important role in its packaging capacity. All of these results lay a basis for researching the relation between the structure and function of alphavirus genome and developing alphavirus vector system with Chinese intellectual property.

关 键 词：XJ-160病毒 复制子载体 定点诱变 

分 类 号：R373.9[医药卫生—病原生物学]