鲤疱疹病毒Ⅱ型ORF25B编码蛋白诱饵载体的构建及其互作蛋白的筛选  

Construction of a bait vector and preliminary screening of host proteins interacting with Cyprinid herpesvirus 2 ORF25B using yeast two hybrid system

作　　者：聂细荣 薛明洋 林格 李逸群 范玉顶[2] 刘文枝[2] 孟彦[2] 江南[2] 曾令兵[2] 周勇[2] Xirong Nie;Mingyang Xue;Ge Lin;Yiqun Li;Yuding Fan;Wenzhi Liu;Yan Meng;Nan Jiang;Lingbing Zeng;Yong Zhou(College of Fisheries and Life Sciences,Shanghai Ocean University,Shanghai 201306,China;Yangtze River Fisheries Research Institute,Chinese Academy of Fishery Science,Wuhan 430223,Hubei Province,China)

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]中国水产科学研究院长江水产研究所,湖北武汉430223

出　　处：《微生物学报》2021年第10期3103-3113,共11页Acta Microbiologica Sinica

基　　金：国家重点研发计划(2019YFD0900105);国家自然科学基金(31802346);国家大宗淡水鱼类产业技术体系建设专项资金(CARS-45-16);湖北省技术创新专项(2018ABA101)。

摘　　要：【目的】鲤疱疹病毒Ⅱ型(Cyprinid herpesvirus 2,CyHV-2)感染养殖鲫引起的鲫造血器官坏死病,给鲫养殖业造成了重大的经济损失。揭示CyHV-2感染宿主细胞的机制,是建立鲫造血器官坏死病有效防治技术的重要基础。【方法】本研究针对CyHV-2富含抗原表位的ORF25B区域设计引物,扩增ORF25B基因截短序列。将扩增产物克隆至酵母双杂交诱饵载体pGBKT7,构建诱饵载体pGBKT7-tORF25B,转化至酵母菌株Y2HGold中。在营养缺陷型培养基上,验证诱饵表达载体pGBKT7-tORF25B对酵母菌Y2HGold自激活现象和毒性作用。利用酵母双杂交技术,将诱饵菌株pGBKT7-tORF25B/Y2HGold与鲫脑组织细胞系(GiCB)cDNA文库杂交。【结果】ORF25B基因截短序列扩增大小约为981 bp,成功构建了诱饵菌株pGBKT7-tORF25B/Y2H Gold,自激活和毒性验证结果表明,诱饵表达载体对酵母菌株无自激活现象,也无毒性作用,初步筛选出4种与tORF25B基因编码蛋白互作的宿主蛋白。【结论】本研究结果为深入开展CyHV-2 ORF25B编码蛋白功能及病毒入侵宿主细胞的机制研究奠定了重要基础。[Objective]The hemopoietic necrosis disease of Carassius auratus gibelio infected by Cyprinid herpesvirus 2(CyHV-2)has caused huge economic losses to the farming industry of this fish.To reveal the mechanism of CyHV-2 infecting host cells might provide an important basis for the investigations of prevention and control technology for this disease.[Methods]In this study,we designed primers for the epitope rich region of CyHV-2 ORF25 B,and amplified truncated ORF25 B gene by polymerase chain reaction(PCR).Next,we cloned the amplified product into yeast two hybrid bait expression vector pGBKT7.Then the bait recombinant vector pGBKT7-tORF25 B was constructed and we transformed it into yeast Y2 H Gold.In addition,we detected the transcriptional self-activation and toxicity of the bait protein on the auxotroph medium.Using yeast two hybrid technique,we hybridized the bait strain pGBKT7-tORF25 B/Y2 H Gold with GiCB cDNA library.[Results]The truncated ORF25 B gene was about 981 bp.It was suggested that the bait strain pGBKT7-tORF25 B/Y2 H Gold was obtained.And the bait vector was shown to have no toxicity to the yeast cells and no self-activation phenomenon to the report genes.There were four candidate host proteins interacting with tORF25 B gene coded protein were preliminarily obtained.[Conclusion]The results of this study have laid a foundation for further study on the protein function of CyHV-2 ORF25 B and the mechanism of this virus invasion into host cells.

关 键 词：鲤疱疹病毒Ⅱ型 ORF25B基因 酵母双杂交 诱饵载体 互作蛋白 

分 类 号：S943[农业科学—水产养殖]