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.
出处
《河南农业科学》
CSCD
北大核心
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)
