应用酵母双杂交系统筛选与流感病毒PB2蛋白相互作用的宿主蛋白  

Identification of host interacting proteins of influenza virus PB2 by yeast two-hybrid system

作　　者：孙楠[1] 梁立滨[1] 王金光[1] 李俊平[1] 罗维玉[1,2] 赵玉辉[1] 朱鹏阳[1] 姜丽[1] 陈化兰[1,2] 李呈军[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/农业部动物流感重点实验室,黑龙江哈尔滨150069 [2]甘肃农业大学动物医学学院,甘肃兰州730070

出　　处：《中国预防兽医学报》2017年第1期15-19,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31472215;31521005)

摘　　要：流感病毒PB2蛋白可以作用于病毒mRNA转录起始阶段,辅助病毒mRNA引物的合成,并能够影响病毒的宿主范围,参与禽流感病毒在哺乳动物宿主体内的适应过程。本研究利用2009甲型H1N1流感病毒PB2蛋白作为"诱饵"蛋白,通过经典酵母双杂交系统筛选由Calu-3、A549、THP-1和U251 4种细胞构建的cDNA文库,筛选到多个与PB2蛋白相互作用的宿主蛋白。针对其中筛选到的一种宿主蛋白FHL2,进行免疫共沉淀(Co-IP)和激光共聚焦(Confocal)试验,证实了PB2蛋白和FHL2蛋白二者之间存在相互作用,并且转染瞬时过表达FHL2发现该蛋白可以负调控流感病毒复制,为进一步研究PB2蛋白与FHL2蛋白互作及影响病毒复制的分子机制奠定了基础。The PB2 protein of influenza virus acts on the initiation stage of viral RNA transcription by assisting the formation of viral mRNA primer. In addition, PB2 protein is involved in the mammalian host adaptation process and has an effect on the host range tropism of influenza virus. In this study, we used a yeast two-hybrid system to screen a cDNA library prepared from a mixture of four human cell lines （A549, Calu-3, THP- and U251） for host proteins that interact with the PB2 protein of 2009 H1N1 influenza virus. One specific interacting partners of PB2, four and a half LIM domains 2 （FHL2）, was selected for further study. The interaction between PB2 and FHL2 was confirmed by performing co-immunoprecipitation and confocal microscopy experiments. We found that overcxpression of FHL2 protein in 293 cells by transient transfection negatively regulated the replication of influenza virus. This study laid a foundation to fiLrther reveal the role of FHL2 in regulating influenza virus life cycle by interacting with PB2 protein.

关 键 词：流感病毒 PB2蛋白 酵母双杂交 免疫共沉淀 激光共聚焦 FHL2 

分 类 号：S852.65[农业科学—基础兽医学]