猪流行性腹泻病毒N蛋白核仁定位信号对宿主细胞周期的影响

Effects on host cell cycle by nucleoli localization signal peptides of porcine epidemic diarrhea virus nucleocapsid protein

为研究猪流行性腹泻病毒(PEDV)核衣壳蛋白(N)与宿主细胞相互作用,本研究根据前期已经鉴定的PEDV N蛋白核仁定位信号序列,利用重叠延伸PCR(SOE-PCR)方法扩增出核仁定位信号序列缺失的PEDV N基因(dN),并将其与真核表达载体pAc-GFP-C1连接,构建重组质粒pAc-GFP-dN。将pAc-GFP-dN转染于Vero E6细胞中,利用激光共聚焦显微镜分析重组质粒pAc-GFP-dN表达蛋白的定位情况,并利用流式细胞仪检测转染细胞的周期变化。实验结果表明:与完整的N蛋白相比,缺失核仁定位信号的N蛋白不能定位于细胞核仁,同时丧失了使宿主细胞周期在G2/M期停滞的能力,从而揭示了N蛋白核仁定位信号对宿主细胞具有重要的影响。

To study the interaction between porcine epidemic diarrhea virus （PEDV） N protein and the host cell, the PEDV N gene was amplified with the deletion of nucleoli localization signal sequence by SOE-PCR and cloned into eukaryotic expression vector pAc-GFP-C 1 to construct the recombinant plasmid pAc-GFP-dN. Veto E6 cells were transfected with the pAc-GFP-dN and the subcellular localization of the expressed dN protein was analyzed by confocal microscopy. The effect on cell cycle of the transfected cell was detected by flow cytometry. The results showed that the dN protein was unable to localize in the nucleus compared to the N protein as expected; However, the dN was also unable to induce the arresting on cell cycle G2/M caused by N orotein. These results revealed that the nucleoli localization signal in N orotein has an important function to host cell.