猪流行性腹泻病毒核衣壳蛋白与感染细胞核磷蛋白的共定位分析

Co-localization analysis between porcine epidemic diarrhea virus nucleocapsid protein and nucleolar phosphoprotein B23. 1

【目的】阐明猪流行性腹泻病毒(PEDV)核衣壳蛋白与病毒感染细胞核仁成分B23.1蛋白的共定位特征。【方法】分别参照GenBank中PEDV CV777株的N基因序列(AF353511)和编码人细胞核仁蛋白B23.1基因序列(BC050628.1),设计、合成扩增N基因和B23.1基因的引物,利用RT-PCR技术扩增了N基因和Vero E6细胞的B23.1基因的cDNA,分别克隆到真核表达载体pAcGFP1-C1和pDsRed2-N1,获得重组质粒pAcGFP1-C1/N和pDsRed2-N1/B23.1,共转染Vero E6细胞。【结果】Western blots分析表明这些融合蛋白在转染的Vero E6细胞中表达;共聚焦显微镜技术分析表明在共转染Vero E6细胞中猪流行性腹泻病毒N蛋白与Vero E6细胞核磷蛋白B23.1发生共定位。【结论】为进一步鉴定PEDV N蛋白中核仁定位信号和N蛋白核仁定位机制提供可靠依据。

[Objective]To elucidate the co-localization characteristic between porcine epidemic diarrhea virus（PEDV） N protein and B23.1 phosphoprotein.[Methods]Two pairs of primers used to amplify N gene and B23.1 gene were designed and synthesized according to CV777 N gene sequence（AF353511） and human nucleolar phosphoprotein B23.1 gene sequence（BC050628.1）,respectively.The PEDV N gene and B23.1 gene were amplified by RT-PCR from PEDV strain CV777 and Vero E6 cells,respectively;then cloned into eukaryotic expression vector pAcGFP1-C1 and pDsRed2-N1,to generate the recombinant plasmids pAcGFP1-C1 / N and pDsRed2-N1 /B23.1,respectively.Vero E6 cells were transfected with plasmids pAcGFP1-C1 / N and pDsRed2-N1 /B23.1.[Results]The fusion proteins successfully expressed in transfected Vero E6 cells by western blot analysis,and the PEDV N protein and the B23.1 phosphoprotein showed co-localization features in co-transfected cells through confocal microscopy analysis.[Conclusion]The results will help to identify the nucleolar localization signals in PEDV N protein and to elucidate the mechanism of N protein located in nucleus.