H3N2亚型猪流感病毒NS2基因表达产物特性分析  

Characteristic analysis of NS2 gene product of swine influenza virus H3N2 subtype

作　　者：郭林[1,2] 王晓杜[1] 刘庆伟[1] 沈阳[1] 邱亚峰[1] 李向东[1] 罗满林[2] 马志永[1] 

机构地区：[1]中国农业科学院上海兽医研究所兽医公共卫生研究室,上海200241 [2]华南农业大学兽医学院,广东广州510642

出　　处：《畜牧与兽医》2014年第9期5-10,共6页Animal Husbandry & Veterinary Medicine

基　　金：上海市浦江人才计划资助(07pj14109);中央级公益性科研院所基本科研业务费专项资金重点项目

摘　　要：采用RT-PCR方法克隆猪流感病毒H3N2亚型NS2全长基因,构建NS2基因原核表达质粒pET-28a-NS2和真核表达质粒p3xFLAG-CMVNS2,在大肠杆菌和真核细胞内表达NS2基因,并制备抗NS2多克隆抗体。用所制备的抗体分析p3xFLAG-CMV-NS2转染表达NS2蛋白和病毒感染细胞内的NS2蛋白。结果表明:经终浓度为1 mmol/L的IPTG诱导后,重组蛋白NS2在大肠杆菌中得到表达。表达蛋白纯化后免疫Wistar大鼠制备抗NS2蛋白多克隆抗体,Western-blot分析表明抗NS2多克隆抗体可以识别大肠杆菌表达的NS2蛋白、转染Vero细胞表达的NS2蛋白和病毒感染细胞内的NS2蛋白。间接免疫荧光发现NS2蛋白主要定位于细胞质。本试验为进一步研究NS2蛋白在病毒复制过程中的生物学功能和猪流感病毒的复制机理奠定基础。To construct the recombinant plasmid carrying NS2 gene of swine influenza virus （SIV） for prokaryotic and eukaryotic expres- sion and to analyze the characteristics of NS2 gene product, we amplified NS2 gene of SIV H3N2 subtype by RT-PCR using genomic RNAsextracted from the allantoic fluid of SIV-infected chicken embryo as template. The amplified product was cloned into the expression vectors pET-28a （＋） and p3xFLAG-CMV-7.1 to construct the recombinant plasmids pET-28a-NS2 and p3xFLAG-CMV-NS2, respectively. Af- ter NS2 was induced to express and purified, the polyclonal antibody against this protein was prepared. Western-blot analysis showed that the produced antibody was able to react with NS2 proteins expressed in Escherichia coli as well as synthesized in SIV-infected cells. The NS2 proteins were predominantly localized in the cytoplasm as detected by immunofluorescence analysis. This study lays the foundation for further study on replication mechanism and biological function of SIV during virus replication.

关 键 词：猪流感病毒 NS2 克隆 表达 特性分析 

分 类 号：S852.65[农业科学—基础兽医学]