草鱼呼肠孤病毒GCRV VP56相互作用蛋白的鉴定

为研究Ⅱ型草鱼呼肠孤病毒外纤维蛋白VP56与宿主蛋白的相互作用,实验采用酵母双杂交Gal4系统鉴定与VP56相互作用的草鱼蛋白。首先利用RT-PCR技术从Ⅱ型GCRV感染的草鱼肾细胞中扩增目的基因VP56,构建p GBKT7-VP56诱饵表达载体;在酵母菌株中检测其自激活性后,以VP56为诱饵在草鱼酵母双杂交文库中筛选阳性菌株,并对阳性克隆的序列进行分析。实验也克隆了草鱼JAM-A(Gc JAM-A)基因,并构建p GADT7-Gc JAM-A载体,在酵母中研究草鱼Gc JAM-A与病毒蛋白VP56相互作用的可能性。研究表明构建的诱饵质粒p GBKT7-VP56无自激活作用,可以应用于酵母双杂交筛选;从草鱼肾细胞文库中筛选得到9株阳性克隆,基因测序及序列分析确定其中包含草鱼7个细胞内蛋白和1个细胞外基质蛋白;VP56蛋白与Gc JAM-A蛋白在酵母中不能发生相互作用。本研究初步确定了与Ⅱ型GCRV蛋白VP56存在潜在相互作用的宿主蛋白,为深入探讨VP56蛋白在Ⅱ型GCRV感染宿主过程中的生物学功能奠定了基础。

Screening of Potential Host Proteins Interacting with VP56 of Type II Grass Carp Reovirus by Yeast Two-Hybrid System

Among the three genotypes of grass carp reovirus (GCRV), type II GCRV accounts for the major epidemic of grass carp hemorrhagic disease at present in China. Bioinformatic analysis predicted that VP56 of type II GCRV might function as the fiber-like protein of reoviruses. This study aimed to screen potential host proteins that interact with VP56, which might present clues on the biological role of VP56. VP56 gene was amplified by RT-PCR and was used to generate the bait plasmid, pGBKT7-VP56. Subsequently, cDNA library plasmid of Ctenopharyngodon idella kidney cells was screened to test potential interaction partners in yeast AH109 transformed with pGBKT7-VP56. The positive clones were analyzed by plasmid sequencing and nucleotide sequence blasting. To test whether JAM-A served as a potential interaction partner for reoviral fiber protein in grass carp like in mammalian cells, pGADT7-GcJAM-A vector was constructed for the sake of further validation of its interaction with VP56 protein. Results in this study showed that the bait plasmid pGBKT7-VP56 has no selfactivation in yeast, 9 positive clones were obtained, and 7 cellular proteins and an extracellular matrix protein were suggested to bind VP56, and no interaction between VP56 protein and GcJAM-A protein was detected in yeast cell. These results may pave the way for further functional analysis of VP56 protein.