A型流感病毒NS1蛋白单克隆抗体的制备及其亚细胞定位分析  

Preparation of monoclonal antibodies against NS1 protein of influenza A virus and analysis of its subcellular localization

作　　者：杨辉[1] 吴小鹏[1] 王海龙[1] 石婷婷[1] 王星博[1] 袁庄川 颜焰[1] 廖敏[1] 周继勇[1] 

机构地区：[1]浙江大学农业部动物病毒学重点实验室,浙江杭州310058

出　　处：《中国兽医科学》2016年第8期953-958,共6页Chinese Veterinary Science

基　　金：国家科技支撑计划重点项目(2015BAD12B01)

摘　　要：以A型流感病毒A/Swine/Guangdong/(H3N2,H3病毒)株的基因组作为模板,扩增NS1基因并构建原核表达载体p ET-28a(+)-NS1。以大肠杆菌表达的NS1蛋白为抗原免疫小鼠,制备单克隆抗体,获得了2株分泌单克隆抗体细胞株2H9和3A4。IFA和Western-blot检测显示,抗NS1单克隆抗体与H3病毒感染的细胞有良好的反应性和特异性。亚细胞定位分析显示,H3N2病毒的NS1聚集于核仁处,H1N1病毒NS1聚集于核内周边,PR8毒株的NS1少部分在核内聚集、多数胞质呈颗粒状分布。流感病毒NS1单克隆抗体的制备为流感病毒流行病学监测、鉴别诊断及对其蛋白功能研究提供了工具。Nonstructure protein I （NSI） gene of influenza virus was amplified by RT-PCR with RNA of A/Swine/Guangdong/（H3N2, H3 virus）strain as template, which were constructed into prokaryotic expression vector of pET-28（＋）,named pET-28（＋）-NSl, and expressed in - coJi. The monoclonal antibodies（mAbs） against NSI were prepared with BALB/c mice immunized with purified recombinant NSI and two hybridoma cell strains （2H9and 3A4） which secreted antibodies against NSl were obtained. The two mAbs showed specific reactions with H3 infected cells as detected by indirect immunofluorescence（IFA） and Western-blot. Subcellular localization analysis showed that H3N2 NSI accumulated in the nucleolus, HiNl NSI gathered in the nuclear periphery, and while some of the PR8 NS1 localized to the nucleus, a significant proportion of PR8NSI was granular widely distributed in the cytoplasm, ln this study, the monoclonal antibodies against NSI were successfully prepared, which might provide an efficient tool for influenza virus detection and further study of the functions of NSI.

关 键 词：流感病毒 NS1 单克隆抗体 亚细胞定位 

分 类 号：S852.659.6[农业科学—基础兽医学]