猪瘟病毒NS3蛋白在真核细胞中的表达与定位

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。

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

To investigate the interaction cellular proteins with swine fever virus NS3 protein, NS3 gene 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 293T cells, 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 293T cells. This study lays a foundation for further verifying the interaction cellular proteins with swine fever virus NS3 protein.