流感病毒NS1蛋白与宿主蛋白DOK6的相互作用研究  

Study on interaction between influenza virus NS1 protein and host protein DOK6

作　　者：黄山雨 梁立滨[1] 李俊平[1] 王倩[1] 赵青青[1] 周陈陈 赵玉辉[1] 王广文[1] 李奇兵 孔凡迪 李呈军[1] 陈化兰[1] 姜丽[1] HUANG Shan-yu;LIANG Li-bin;LI Jun-ping;WANG Qian;ZHAO Qing-qing;ZHOU Chen-chen;ZHAO Yu-hui;WANG Guang-wen;LI Qi-bing;KONG Fan-di;LI Cheng-jun;CHEN Hua-lan;JIANG Li(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

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

出　　处：《中国预防兽医学报》2019年第10期987-992,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31402206、31672582)

摘　　要：A型流感病毒非结构蛋白1(NS1)是病毒编码的必需蛋白,在病毒复制周期中发挥重要作用。NS1蛋白在病毒侵入宿主后大量表达,抑制宿主干扰素通路的激活,选择性关闭宿主基因的表达且能够增强病毒蛋白的表达。为研究宿主DOK6蛋白与NS1蛋白的相互作用关系,本研究利用重组质粒p CAGGS-V5-DOK6、p CAGGS-Flag-NS1共转染293T细胞,利用免疫共沉淀试验(Co-IP)检测,结果显示沉淀NS1蛋白后能够检测到DOK6蛋白条带,表明DOK6蛋白与NS1蛋白存在相互作用;将二者共转染A549细胞后利用激光共聚焦显微镜观察,发现二者在细胞质中存在共定位,表明两种蛋白在细胞质中发生相互作用。通过双分子荧光互补试验(BiFC)检测,结果显示NS1蛋白能够与DOK6蛋白的磷酸化酪氨酸结合结构域(Phosphotyrosine binding domain,PTB)结合从而发出较强荧光,表明NS1蛋白能够与DOK6蛋白(PTB)结构域发生相互作用。本研究进一步完善了A型流感病毒NS1蛋白参与的复杂分子间相互作用网络,为深入理解流感病毒在宿主体内复制的分子调控机制奠定了基础。Non-structural protein 1(NS1)is one of the essential viral proteins encoded by influenza A virus,playing an important role in the virus replication cycle.NS1 protein is abundantly expressed in the virus infection cycle,inhibiting the activation of the host interferon pathway.It can also selectively reduce the expression of the host proteins so as to enhance the expression of the viral proteins.To study the interaction relationship between host DOK6 protein and NS1 protein,the eukaryotic expression vectors pCAGGS-V5-DOK6 and pCAGGS-Flag-NS1 were co-transfected into 293 T cells and their interaction was detected by co-IP assay.DOK6 was detected by western blotting after immunoprecipitated by the NS1 protein,indicating that DOK6 protein interacted with NS1 protein.The expression vectors were then co-transfected intoA549 cells,and we found that the two proteins co-localized in the cytoplasmby confocal microscopy assay,which indicated that they interacted in the cytoplasm.By use of Bi FC assay,we also detected strong fluorescence when NS1 and the PTB domain of DOK6 were co-expressed,showing that NS1 protein interacted with the PTB domain of DOK6.Our study enriched the interaction network between influenza virus NS1 and host proteins,thus facilitating the better understanding of the mechanismof the influenza virus replication in the host.

关 键 词：流感病毒 NS1蛋白 DOK6蛋白 蛋白相互作用 

分 类 号：S852.65[农业科学—基础兽医学]