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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。
In order to observe the influence of nsP2-726Pro site-directed mutagenesis on the characteristics of XJ-160 replicon vector of Sindbis virus (SINV), the mutant vector was constructed by using site-directed mutagenesis using pBRepXJ as the molecular basis of XJ-160 virus replicon vector pBRep-726L, pBRep-726S, pBRep-726V and pBRep-726A. The neomycin resistance gene (Neomycin) was cloned into pBRepXJ and its mutants. The effect of nsP2-726Pro site-directed mutagenesis on Cytopathic effect (CPE) was observed by cell culture method. EGFR and R. luc gene were inserted into each vector respectively, and the results of site-directed mutagenesis were qualitatively and quantitatively examined. The replicon vector autonomously replicated The impact of ability. The results showed that the ability of pBRep-726V and pBRep-726A to replicate autonomously in BHK-21 cells was higher than that of pBRepXJ, and the induced cytopathic effect was faster. The introduction of the substitution mutation nsP2-726Pro → Leu completely loses the ability of the vector to cause CPE on the cells while maintaining the packaging ability. While pBRep-726S has an intermediate phenotype. Tip nsP2-726Pro site-directed mutagenesis in the XJ-160 replicon autonomously replicating capacity, but also changed the CPE process. This will lay the foundation for the study of the relationship between Sindbis virus genomic structure and function and construction of non-cytopathic alphavirus vector.