应用杆状病毒系统高效表达猪繁殖与呼吸综合征病毒GP5基因

将猪繁殖与呼吸综合征病毒CH_la株囊膜糖蛋白 (GP5 )基因定向亚克隆到杆状病毒转移载体pBlueBacHisB中 ,与杆状病毒线性DNA(Bac_N_BlueTMDNA)共转染昆虫细胞sf9,经过三轮蚀斑纯化后 ,获得重组杆状病毒rBac_GP5。用该重组病毒感染sf9细胞后 ,应用间接免疫荧光试验、SDS_PAGE和Westernblot检测表明 ,GP5基因在杆状病毒系统中获得了高效表达 ,表达产物因糖基化程度差异 ,表观分子量有 2 2、2 5和 30kDa三种总计约占细胞蛋白总量的 2 6 .4%。

High Level Expression of Porcine Reproductive and Respiratory Syndrome Virus GP5 Gene in Baculovirus System

Porcine reproductive and respiratory syndrome virus(PRRSV)strain CH-la,a Chinese reference strain,was isloated in Beijing suburb in themiddle 1990 Its GP5 gene encoded by open reading frame(ORF)5 was cloned and inserted intothe baculovirus transfer vector pBlueBacHis B. Sf9 cells were co-transfected by therecombinant transfer plasmid and linear baculovirus DNA.A r ecombinant baculovirus wasobtained after blue-plaque screening and three cycles of plaque-purification.The GP5 genewas confirmed to be expressed in sf9 cells by immunofluorescent staining and Westernblot.The expressed recombinant fusion protein with Histag have apparent molecular weightof 22,25,and 30,accounting fo r 26.4% approximately in whole cellular proteins.