禽呼肠孤病毒蛋白p17互作蛋白的筛选与鉴定

Screening and Identification of the Protein P17 Interaction Proteins of Avian Reovirus

禽呼肠孤病毒(ARV)p17蛋白在诱导细胞自噬和促进病毒复制中发挥重要作用,但与宿主细胞之间的相互关系及其致病机制尚不清楚。本研究将ARV GX/2010/1株病毒液以10^(4.3)TCID50/0.2 mL通过滴鼻点眼注射感染7日龄SPF鸡。应用Smart^(TM)技术构建该鸡肝组织cDNA文库,且文库滴度达到2.6×10^(7)CFU/mL。以p17为诱饵蛋白,利用酵母双杂交系统筛选得到16个互作候选克隆,经酵母回复验证得到9个阳性克隆并送至测序。通过NCBI和Uniport等在线数据库比对分析得到PRPS2、IFI16、GTPBP4、IGF2BP1等胞内互作蛋白。基因功能分析显示,这些蛋白参与宿主细胞先天免疫反应、RNA的运输与翻译、结合核糖蛋白复合物、细胞粘附与迁移等生物学过程。对这些互作蛋白的深入研究将为进一步了解ARV及其p17蛋白致病的分子机制奠定基础。

Previous studies have shown that the Avian Reovirus(ARV)p17 protein plays an important role in inducing cell autophagy and promoting virus replication.However,its interaction with host cells and pathogenic mechanism are still unclear.In this study,ARV GX/2010/1 virus solution was injected into 7-day-old SPF chickens with 10^(4.3)TCID50/0.2 mL through nasal dripping.The Smart^(TM)technology was used to construct the chicken liver tissue cDNA library,which was determined to the titer of 2.6×10^(7)CFU/mL.Using p17 as the bait protein,16 candidate clones related to interaction were screened using the yeast two-hybrid system,and 9 positive clones were obtained by yeast response verification and sequenced.Intracellular interacting proteins such as PRPS2,IFI16,GTPBP4,and IGF2BP1 were obtained through analysis of online databases such as NCBI and Uniport.Gene function analysis showed that these proteins were involved in biological processes such as host cell innate immune response,RNA transport and translation,binding to riboprotein complexes,cell adhesion and migration.The in-depth study of these interacting proteins would lay the foundation for further investigation of the molecular mechanism of ARV and its p17 protein.