猪繁殖与呼吸综合征病毒GP5蛋白在酿酒酵母中的定位研究

为研究猪繁殖与呼吸综合征病毒(PRRSV)gp5基因在酿酒酵母中表达后蛋白的定位位置,以此揭示PRRSV在感染细胞中获取囊膜的位置信息,本研究首先将gp5基因克隆到酿酒酵母表达载体pUG35的gfp基因上游,构建重组质粒pUG-gp5-gfp,电击导入酿酒酵母CEN.PK2中,经尿嘧啶缺陷型营养筛选,获得重组酵母pUG-gp5-gfp/CEN.PK2,在甲硫氨酸营养缺陷培养基诱导表达后,荧光显微镜观察显示,gp5-gfp融合基因在酿酒酵母中获得表达,所表达的融合荧光蛋白主要聚积位置与内质网和质膜的定位模式一致,表明GP5蛋白信号肽在酵母中起到了导向作用,揭示了PRRSV在宿主细胞中获得囊膜的可能位置。该研究为其他病毒在宿主细胞中的组装位置研究提供新的思路。

Membrane localization of PRRSV GP5 protein in Saccharomyces cerevisiae

In porcine reproductive and respiratory syndrome virus(PRRSV) infected cells,the envelope protein of GP5 was synthesized and transported to the host cell membrane before PRRSV assembling.To investigate the membrane localization character of PRRSV GP5 protein expressed in Saccharomyces cerevisiae as cell model,the GP5 gene was amplified from PRRSV and inserted into plasmid pUG35 in upstream of GFP gene to construct the recombinant plasmid of pUG35-gp5-gfp,which was transformed into S.cerevisiae by electroporation.The recombinant yeast was cultured with methionine deficient medium,and the fluorescence examination indicated that the GP5-GFP fusion proteins were mainly localized on endocytoplasmic reticulum and cytoplasm of yeast cells.These results demonstrated that the signal peptide of GP5 protein had the ability to direct GP5 protein to cell membranes,from where the envelope of PRRSV were obtained at the time it forming intact virus.