甲型H7N9流感病毒NS1蛋白真核表达载体的构建与表达  

Construction and Expression of NS1 Protein Eukaryotic Expression Vector of Influenza A Virus Subtype H7N9

作　　者：武立伟[1] 李霆[1] 杨世梅[2] 王慧煜[1] 梅琳[1] 韩雪清[1] 

机构地区：[1]中国检验检疫科学研究院,北京100029 [2]西北民族大学,甘肃兰州730124

出　　处：《中国畜牧兽医》2014年第8期66-70,共5页China Animal Husbandry & Veterinary Medicine

基　　金：国家重点基础研究发展计划资助项目(973项目)流感等病毒在不同宿主中的复制机制(2011CB504704-X);基本科研业务费H7N9流感病毒非结构蛋白NS1的功能研究(2014JK007)

摘　　要：本试验旨在构建甲型H7N9流感病毒非结构蛋白(NS1)真核表达载体,并在293T细胞中表达其编码的NS1蛋白。首先提取安徽分离株甲型H7N9流感病毒(A/Anhui/3/2013(H7N9))的总RNA,通过RT-PCR技术获得了甲型H7N9流感病毒H7N9 NS1全长基因,然后将其克隆至载体pcDNA4-Flag-HA中构建pcDNA4-Flag-HA-NS1真核重组表达载体,经酶切及测序鉴定正确后将质粒pcDNA4-Flag-HA-NS1转染到293T细胞中,通过Western blotting鉴定NS1蛋白的表达。结果表明成功克隆了NS1全长基因,构建了甲型H7N9流感病毒NS1蛋白真核表达载pcDNA4-Flag-HA-NS1,并在293T细胞中转染表达,Western blotting确定了NS1蛋白的成功表达。该表达载体的成功构建及在293T细胞中成功表达NS1蛋白,为后期开展流感病毒NS1蛋白功能及与真核细胞中的蛋白相互作用奠定了基础。This study was designed to construct non-structural protein NS1 eukaryotic expression vector of influenza A virus subtype H7N9 and express the NS1 protein in 293 Tcells.The NS1 gene of influenza A virus subtype H7N9 was amplified by RT-PCR and cloned into pcDNA4-Flag-HA vector to construct a plasmid,named pcDNA4-Flag-HA-NS1.The recombinant eukaryotic expression vector pcDNA4-Flag-HA-NS1 subsequently yielded.The expression of the NS1 gene in transfected 293 T cells was tested by Western blotting.The results showed that the recombinant eukaryotic expression vector pcDNA4-Flag-HANS1 was successfully constructed;the NS1 protein was finally expressed in 293T cells;the full-length NS1 gene was obtained as well as its recombinant eukaryotic expression plasmid was successfully constructed and expressed in 293 Tcells.The construction of eukaryotic expression plasmid of NS1 gene was made,and made foundations for further study the function of NS1 protein and the mechanism of diseases induced by influenza A virus.

关 键 词：甲型H7N9流感病毒 NS1 真核表达 Western BLOTTING 

分 类 号：R373[医药卫生—病原生物学]