流感病毒PA蛋白与宿主蛋白PCBP1的相互作用  

Interaction between Influenza Virus PA Protein and Host Protein PCBP1

作　　者：赵青青[1] 李俊平[1] 梁立滨[1] 黄山雨 周陈陈 赵玉辉[1] 王倩[1] 周圆[1] 姜丽[1] 陈化兰[1] 李呈军[1] ZHAO QingQing, LI JunPing, LIANG LiBin, HUANG ShanYu, ZHOU ChenChen, ZHAO YuHui, WANG Qian, ZHOU Yuan,JIANG Li, CHEN HuaLan, LI ChengJun(State Key Laboratory of Veterinary Biotechnology/Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences/Animal Influenza Key Laboratory of the Ministry of Agriculture, Harbin 15006)

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开放实验室,哈尔滨150069

出　　处：《中国农业科学》2018年第17期3389-3396,共8页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31472215;31602077);中国博士后科学基金(2016M601194)

摘　　要：【背景】PA蛋白是流感病毒RNA聚合酶复合体的重要组成部分,在流感病毒基因组的转录和复制过程中发挥重要作用。前期利用酵母双杂交技术(Y2H)筛选与流感病毒PA蛋白相互作用的宿主蛋白,获得多聚胞嘧啶结合蛋白1(poly(r C)-binding protein 1,PCBP1)。【目的】探索PCBP1蛋白与流感病毒PA蛋白的互作关系及其对流感病毒复制的影响,为深入理解流感病毒在宿主体内的复制调控机制提供数据。【方法】将诱饵质粒pGBKT7-PA与筛选到的重组质粒p GADT7-PCBP1以及阴性对照组和阳性对照组按照醋酸锂法分别共转化酵母感受态细胞,并涂布在3种营养缺陷型培养基上,30℃倒置培养5—7 d,观察酵母菌落生长情况和菌落颜色,回交验证PA蛋白与PCBP1蛋白在酵母系统中的相互作用。参照GenBank中录入的PA蛋白和人源PCBP1蛋白的序列,分别设计特异性扩增引物,构建真核重组表达质粒pCAGGS-Flag-PA和pCAGGS-Myc-PCBP1,将这两种真核表达质粒分别单独转染或共同转染HEK293T细胞,于转染48 h后裂解细胞,收获上清,留取少部分作对照,余下的样品逐步加入FLAG单抗和Protein G琼脂糖珠子进行免疫共沉淀(Co-IP),经SDS-PAGE和Western blot检测PA蛋白与PCBP1蛋白在哺乳动物细胞中的相互作用。利用慢病毒包装系统pLVX-IRES-Zs Green1在HEK293T细胞中包装假病毒,将假病毒侵染A549细胞后经超速流式分选构建PCBP1蛋白过表达细胞系,Western blot检测PCBP1过表达情况,然后用流感病毒WSN以0.01 MOI感染该过表达细胞系,于感染后24和48 h收获上清,对上清中的流感病毒进行蚀斑计数。合成PCBP1蛋白的si RNA,转染A549细胞下调PCBP1蛋白的表达,干扰后48 h通过Western blot检测PCBP1蛋白表达下调情况,并以MOI=0.01感染流感病毒WSN,收获感染后24和48 h的上清,进行蚀斑滴定计数。【结果】通过酵母回交验证发现诱饵质粒pGBKT7-PA与重组阳性质粒p GADT7-PCBP1的共转酵母�【Background】The PA protein is an integral component of the influenza virus RNA polymerase complex and plays an important role in the transcription and replication of the viral genome. The yeast two-hybrid system was used in our laboratory to screen for host proteins that interact with PA protein, and one of them was identified to be poly（r C）-binding protein 1（PCBP1）. 【Objective】This study investigated the interaction between PCBP1 protein and influenza virus PA protein and the role of PCBP1 on the replication of influenza virus, which would provide scientific data for the in-depth understanding of the replication regulation mechanism of influenza virus in the host. 【Method】In order to verify the interaction between PA protein and PCBP1 protein in yeast system, the bait plasmid pGBKT7-PA and the selected prey plasmid p GADT7-PCBP1 as well as the negative control and positive control were cotransformed into yeast competent cells, respectively, according to the Li Ac transformation procedure. The transformed yeasts were then spread on three kinds of auxotrophic medium and incubated at 30℃ for 5-7 days to observe the growth and color of colonies. According to the sequences of PA protein and human PCBP1 protein registered in the GenBank, specific amplification primers were designed to construct eukaryotic recombinant expression plasmids pCAGGS-Flag-PA and pCAGGS-Myc-PCBP1. These two eukaryotic expression plasmids were individually transfected or co-transfected into HEK293 T cells. The transfected cells were lysed 48 hours after transfection to harvest the supernatant. A small portion of the supernatant was used as a control, and the rest of supernatant was successively added with the FLAG monoclonal antibody and Protein G agarose beads for the preparation of immunoprecipitates. Protein samples were analyzed by SDS-PAGE and Western blot to detect the interaction between PA protein and PCBP1 protein in mammalian cells. To establish a PCBP1-overexpressing cell line, the pseudovirus was packaged in

关 键 词：酵母双杂交 流感病毒 PA蛋白 PCBP1蛋白 蛋白互作 

分 类 号：S852.65[农业科学—基础兽医学]