流行性腹泻病毒M基因与甲病毒载体重组RNA的构建

Construction of Recombinant RNA of PEDV M Gene and Alphavirus Vector

将猪流行性腹泻病毒(PEDV)的M基因导入甲病毒载体(plasmid Semliki ForestViru,pSFV) ,研究该基因及其编码蛋白的表达。首先将扩增的病毒M 基因导入pSFV的SP6 启动子下游。用Spe Ⅰ酶将pSFV M重组质粒DNA 线性化,再将其体外转录成5′末端含帽子结构的重组RNA。然后通过电穿孔法将此重组RNA转染BHK 21 细胞,收集病毒样粒子用于感染BHK 21 细胞。采用细胞免疫化学的方法对转染和感染细胞的表达产物进行分析。pSFV M RNA 转染和病毒粒子感染哺乳动物细胞表达的M 蛋白,可与PEDV多克隆抗体起特异反应,证明体外转录的重组RNA 含有PEDV M的特异序列。

The amplified M gene of Porcine epidemic diahrrea virus was cloned into the downstream SP6 promoter of pSFV vector. pSFV-M DNA and helper DNA linearized by the enzyme Spe I digestion were both transcribed in vitro into recombinant RNAs which contained the capping analog on the 5′terminal. The recombinant virus particles were obtained with recombinant RNA and helper RNA cotransfected into BHK21 cells by electroporation.And then the recombinant virus particles infected into BHK21 cells. The expressed products were detected by cell immunochemistry. The M protein expressed in transfected cells or infected cells reacted specifically with polyclonal antibody against PEDV. It was demonstrated that the in vitro transcribed recombinant RNA contains specific M sequence of PEDV.