猪瘟病毒NS3蛋白在真核细胞中的表达与定位

Expression and Location of Classical Swine Fever Virus NS3 Protein in Eukaryotic Cells

为研究与猪瘟病毒NS3蛋白相互作用的宿主细胞蛋白,采用PCR方法获得NS3基因,将其定向克隆至pCMV-Myc真核表达载体,经酶切鉴定及序列测定分析,将鉴定为阳性重组表达质粒命名为pMycNS3。采用脂质体介导法将pMyc-NS3转染至PK-15细胞和293T细胞,Western blot和间接免疫荧光检测目的蛋白的表达。结果表明,成功构建重组表达质粒pMyc-NS3,Western blot表明,NS3蛋白在PK-15细胞和293T细胞中均得到表达,NS3在PK-15和293T细胞质中呈弥散性分布。说明,成功构建的猪瘟病毒NS3基因与Myc融合表达质粒,为验证NS3蛋白与宿主细胞蛋白相互作用奠定基础。

To investigate the interaction cellular proteins with swine fever virus NS3 protein,NS3gene was amplified by PCR,then subcloned into pCMV-Myc to construct the expression vector according to the reading frame,confirmed by sequencing and restriction digestion,the positive recombinant plasmid named pMyc-NS3.Using Lipofectamine 2000,the pMyc-NS3 were successfully transformed into PK-15 and 293Tcells,respectively.The expression of NS3 gene was analyzed by Western blot and immunofluorescence assay.The expressed CSFV NS3 protein predominantly localized in the cytoplasma.The recombinant plasmid for co-expression CSFV NS3 and Myc was successfully constructed and expressed in PK-15 and 293Tcells.This study lays a foundation for further verifying the interaction cellular proteins with swine fever virus NS3 protein.