单纯疱疹病毒Ⅱ型多功能蛋白ICP27在Vero细胞中的表达及定位

Expression and Subcellular localization of ICP27 in Vero Cells

目的 构建单纯疱疹病毒Ⅱ型（Herpes simplex virus type 2,HSV-2）感染细胞蛋白27（infected-cell protein 27,ICP27）在Vero细胞中的表达载体,并对其表达进行亚细胞定位.方法 用前期构建成功的pCDNA3.0-ICP27及pEGFP-ICP27通过脂质体介导质粒瞬时转染Vero细胞,经Western blot检测ICP27蛋白的表达.并通过荧光显微镜及免疫组化的方法观察ICP27在Vero细胞中的亚细胞定位.结果 ICP27可在Vero细胞中高表达,pEGFP-ICP27转染Vero细胞12h后,就可以在少量的细胞中表达,而在转染后的24h-48h表达量最大.但转染72h后,表达的蛋白含量在细胞中减少.Western blot结果显示,pEGFP-ICP27和pCDNA3.0-ICP27均可表达ICP27,用质粒pEGFP-ICP27转染的Vero细胞,观察荧光却只发现ICP27定位于细胞核中,而用免疫组化方法检测pCDNA3.0-ICP27发现细胞核和细胞质中均有表达.结论 pEGFP-ICP27和pCDNA3.0-ICP27均可成功的使ICP27在Vero细胞中有效表达,且ICP27在细胞核和细胞质中均有表达,

Objective To construct HSV-2 ICP27 expression vector in Vero cells and to investigate its expression and subcellular localization.Methods The Vero cells were transiently transfected with preconstructed plasmid pCDNA3.0-ICP27 and pEGFP-ICP27.ICP27 protein expression was determined by Western blot.The subcellular localization of ICP27 in Vero cells were analyzed by fluorescence microscopy and immunohistochemical methods.Results ICP27 expression was observed in Vero cells transfected with pEGFP-ICP27 from 12h after transfection,and it was expressed abundantly from 24h to 48h,but decreased after 72h.The result of western blot showed that ICP27can be expressed in the Vero cell transfected with pEGFP-ICP27 or pCDNA3.0-ICP27.Furthermore,ICP27 expression was observed only in nucleus in cell transfected with pEGFP-ICP27 whereas it can be expressed both in nucleus and cytoplasma in cells transfected with pCDNA3.0-ICP27.Conclusions The ICP27 gene in pEGFP-ICP27 and pCDNA3.0-ICP27 could be expressed in Vero cell.Furthermore,ICP27 can be expressed in nucleus and cytoplasm,but in nucleus mainly.