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

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Y2H Gold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Y2H Gold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/Y2H Gold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。

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

Abstract:

[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 ORF25B, and amplified truncated ORF25B 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-tORF25B was constructed and we transformed it into yeast Y2H 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-tORF25B/Y2H Gold with GiCB cDNA library.[Results] The truncated ORF25B gene was about 981 bp. It was suggested that the bait strain pGBKT7-tORF25B/Y2H 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 tORF25B 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 ORF25B and the mechanism of this virus invasion into host cells.

Key words:Cyprinid herpesvirus 2;ORF25B gene;yeast two hybrid;bait vector;interacted protein