猪流行性腹泻病毒N蛋白与Vero E6核仁蛋白Fibrillarin的亚细胞定位分析

Subcellular localization analysis between porcine epidemic diarrhea virus nucleocapsid protein and fibrillarin

为鉴定猪流行性腹泻病毒(PEDV)核衣壳蛋白(N)与Vero E6细胞核仁fibrillarin蛋白的亚细胞定位的特征。本实验参照GenBank中人细胞fibrillarin基因(M59849.1)序列,设计并合成特异性引物,利用RT-PCR方法扩增了Vero E6细胞的fibrillarin基因,并将其克隆于真核表达载体pDsRed2-N1中,构建重组质粒pDsRed-Fib。将pDsRed-Fib与本实验室构建的含有PEDV N基因的重组质粒pAcGFP-N,共转染Vero E6细胞。Western blot结果表明fibrillarin及N融合蛋白在转染的Vero E6细胞中均获得表达;利用共聚焦显微镜观察显示在共转染VeroE6细胞中PEDV N蛋白与Vero E6细胞核仁中的fibrillarin发生共定位。该结果为进一步研究PEDV和核仁蛋白相互作用奠定基础。

To identify the interaction of porcine epidemic diarrhea virus（PEDV） N protein and fibrillarin in Vero E6 cells,the fibrillarin gene were amplified by RT-PCR from Vero E6 cells with a pairs of primers synthesized according to human fibrillarin gene,and cloned into eukaryotic expression vector pDsRed2-N1 to generate the recombinant plasmids pDsRed-Fib.Vero E6 cells were transfected with plasmids pAcGFP-N and pDsRed2-Fib.Western blot results showed that the fusion proteins expressed in transfected Vero E6 cells.Furthermore,the PEDV N protein and the fibrillarin showed co-localization features in nucleolar of co-transfected Vero E6 cells through confocal microscopy observation.The results would be helpful to elucidate the intereaction of PEDV N protein and nucleolar protein.