斑点叉尾病毒囊膜蛋白ORF6在昆虫细胞中的表达  

Expression of channel catfish virus ORF6 gene in insect cells

作　　者：黄锋涛[1] 熊海林[1] 曹胜波[2] 熊传喜[1] 王敏[1] 王卫民[1] 吴兵[1] 刘玉林[1] 刘学芹[1] 

机构地区：[1]华中农业大学水产学院农业动物遗传育种与繁殖教育部重点实验室,湖北武汉430070 [2]华中农业大学动物医学院,湖北武汉430070

出　　处：《水产学报》2010年第7期1034-1039,共6页Journal of Fisheries of China

基　　金：国家自然科学基金项目(30901118);教育部新教师基金项目(20070504047);湖北省自然科学基金项目(2008CBB099);华中农业大学教师启动基金项目(2006XRC047)

摘　　要：为构建基于杆状病毒表达系统的CCV新型亚单位疫苗,将CCV的囊膜蛋白基因ORF6克隆至杆状病毒转座载体pFastBacTM 1质粒中,并将阳性重组转座质粒转化进含穿梭载体Bacmid的感受态细胞DH10Bac中,获得重组子rBacmid-ORF6。在脂质体介导下将该重组子转染sf9昆虫细胞,获得重组杆状病毒AC-ORF6。AC-ORF6感染的sf9昆虫细胞,经超薄切片电镜观察,可见重组杆状病毒呈多粒包埋,经间接免疫荧光、Western-blotting检测,CCV的ORF6蛋白可以在感染了AC-ORF6的sf9细胞中表达。研究表明,获得了插入ORF6基因的重组杆状病毒,并且该基因可以在重组杆状病毒介导下在昆虫细胞中表达,从而为基于CCV ORF6的杆状病毒亚单位疫苗研究奠定了基础。Channel catfish virus（CCV） is a pathogen of channel catfish.This virus can cause severe hemorrhagic disease in young channel catfish and cause major losses to fish farmers.As no efficacious treatment exists,a vaccine against CCV is essential for prevention and control of this disease.Recently,the baculovirus was described as an attractive gene delivery vehicle and a potential vector for vaccine development.In order to express the membrane protein of the CCV ORF6 gene in insect cells,ORF6 gene was subcloned into baculovirus transfer vector（pFastBac^TM1）.The recombinant plasmid pFast-ORF6 was identified by restriction enzyme digestion and gene sequencing.DH10Bac containing baculovirus shutter vector（bacmid） and helper vector was transformed with recombinant plasmid pFast-ORF6.The recombinant bacmid（rBacmid-ORF6） was identified by blue/white selection and PCR analysis.Then the rBacmid-ORF6 was transfected into sf9 cells with Cellfectin Reagent and recombinant baculovirus was obtained in sf9 cells,named AC-ORF6.AC-ORF6 was multiple-nucleopolyhedrovirus which was observed by electron microscopy in the ultra thin section of infected sf9 cells.After AC-ORF6 infection,the recombinant CCV ORF6 protein was characterized by indirect immunofluorescence assay and Western-blotting.The results showed that ORF6 protein was expressed in sf9 cells and had a molecular mass of 16 ku.This study will provide useful basis for the subunit CCV vaccine development in the future.

关 键 词：斑点叉尾鮰病毒 ORF6蛋白 杆状病毒 昆虫细胞 表达 

分 类 号：Q785[生物学—分子生物学] S941.41[农业科学—水产养殖]