H5N1禽流感病毒HA基因的重组腺病毒表达载体的构建  

Construction of A Recombinant Adenovirus Expression Vector Containing the HA Gene of H5N1 Avian Influenza Virus

作　　者：王青[1] 胡建和[1] 徐彦召[2] 朱国坡[1] 郑素玲[1] 

机构地区：[1]河南科技学院动物科学学院,河南新乡453003 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《河南农业科学》2010年第7期92-96,共5页Journal of Henan Agricultural Sciences

基　　金：河南省高校杰出科研人才创新工程项目(2006KYCX020)

摘　　要：采用PCR方法扩增出禽流感病毒HA基因,将其定向插入pAdtrack-CMV腺病毒穿梭质粒中构建pAdtrack-H5,之后将pAdtrack-H5与腺病毒骨架质粒pAdeasy-1在基因工程菌BJ5183中进行同源重组,获得腺病毒质粒pAdeasy-H5,将pAdeasyd-H5经PacⅠ线性化后转染HEK293细胞株,包装出含有HA基因的腺病毒pAd-H5。结果表明:含有目的基因的腺病毒穿梭质粒pAdtrack-H5和含有目的基因的腺病毒质粒pAdeasy-H5经PCR、双酶切及核苷酸测序鉴定无误。包装成功的腺病毒pAd-H5经绿色荧光蛋白和RT-PCR分析证实,目的基因在该细胞中成功表达。In the present work,the HA gene of avian influenza virus strain H5N1 was amplified from the plasmid pMD-19T-H5 by PCR and inserted into pAdtrack-CMV to construct a shuttle plasmid pAdtrack-H5.Then,the shuttle plasmid was transformed into BJ5183,which containing adenovirus backbone vector pAdeasy-1,to obtain recombinant adenovirus genome plasmid named as pAdeasy-H5.Both pAdtrack-H5 and pAdeasy-H5 were further identified by PCR,double-digestion and DNA sequencing.Then,pAdeasy-H5 was linearized with PacⅠ and transfected into HEK293 cells to form pAd-H5 viruses.GFP and RT-PCR were used to detect the expression of HA gene and the results showed HA pAdeasy-H5 was successfully expressed in HEK 293 cells.

关 键 词：腺病毒载体 禽流感病毒 H5N1亚型 HA基因 载体构建 

分 类 号：S852.65[农业科学—基础兽医学]