利用酵母双杂交技术筛选草鱼Ⅲ型呼肠孤病毒VP6和VP38相互作用蛋白的研究

Yeast two-hybrid screening of host proteins that interact with VP6 or VP38 encoded by type Ⅲ reovirus of grass carp

为探究草鱼Ⅲ型呼肠孤病毒VP6和VP38蛋白的生物学功能,利用酵母双杂交Gal4系统分别筛选了能与GCRV104毒株编码的VP6和VP38相互作用的宿主蛋白。利用RT-PCR从感染GCRV104的草鱼肾脏细胞中扩增GCRV104 s8和s10基因组片段的编码基因后,通过酶切与连接分别克隆至载体p GBKT7中,构建诱饵质粒p GBKT7-VP6和p GBKT7-VP38。对这些重组质粒进行细胞毒性和自激活检测后,分别以VP6和VP38为诱饵在草鱼酵母文库中筛选与其相互作用的宿主蛋白,对筛选得到的阳性酵母菌落进行序列分析。结果表明,两个诱饵质粒p GBKT7-VP6和p GBKT7-VP38均无自激活作用;诱饵质粒p GBKT7-VP6筛选到7株阳性克隆,分别编码β肌动蛋白、augmin样复合体亚基2、甘露糖苷酶α2b1亚基、程序性细胞死亡蛋白6、真核翻译延长因子1γ和一个未知功能蛋白;诱饵质粒p GBKT7-VP38筛选到4株阳性克隆,分别编码剪切与多聚腺苷酸化特异性因子5、高迁移率组蛋白核小体结合结构域2、葡萄糖转运体X和蛋白酶体亚基β7。结果为进一步探究GCRV104毒株编码的VP6、VP38与宿主蛋白及其涉及的信号通路的相互作用奠定了基础。

To screen and identify the proteins that interact with VP6 and VP38 of type III reovirus of grass carp,GCRV104 strain, the complete ORFs encoded by the s8 and s10 genome fragments were amplified by RT-PCR from infected cells and used to generate the bait plasmids for yeast two-hybrid screen, pGBKT7-VP6 and pGBKT7-VP38, respectively. The cDNA library plasmid of Ctenopharyngodon idella kidney cells was screened to reveal potential interaction partners in yeast AH109 transformed with pGBKT7-VP6 or pGBKT7-VP38. The positive clones were analyzed by plasmid sequencing and nucleotide sequence blasting.The results showed that both bait plasmid pGBKT7-VP6 and pGBKT7-VP38 demonstrated no self-activation in yeast; 7 host proteins were suggested to bind VP6 including beta-actin, augmin-like complex subunit 2, zinc finger FYVE domain containing 21, mannosidase alpha class 2B member 1, programmed cell death 6, eukaryotic translation elongation factor 1 gamma and an unknown protein; while, 4 host proteins were suggested to bind VP38 that included cleavage and polyadenylation specific factor 5, high mobility group nucleosomal binding domain 2, glucose transporter X （glutX） gene, and proteasome subunit beta7. This study may pave the way for understanding functions of VP6 and VP38 proteins encoded by genotype Ⅲ reovirus of grass carp and present some information for the exploration of GCRV replication mechanism.