摘要
构建携带有H5N1亚型AIV NA基因的重组腺病毒表达载体,为进一步研究AIV中NA基因在病毒致病过程中的作用及相关诊断试剂的开发提供依据。采用PCR的方法从构建好的包含有H5N1AIV NA基因的pMD-19T-N1质粒中扩增出NA基因,定向插入pAdtrack-CMV腺病毒穿梭质粒中,含有目的基因的腺病毒穿梭质粒pAdtrack-N1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-N1,将pAdeasyd-N1经pacⅠ线性化后转染HEK293细胞株,包装出含有NA基因的腺病毒pAd-N1。结果表明,构建含有目的基因的腺病毒穿梭质粒pAdtrack-N1和含有目的基因的腺病毒质粒pAdeasy-N1经PCR、双酶切及核苷酸测序测定无误。线性化后的pAdeasy-N1转染HEK293细胞,成功获得腺病毒pAd-N1载体,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。
NA gene was amplified from the pMD-19T-N1 by PCR, and cloned into shuttle plasmid pAdTraek-CMV. The shuttle plasmid was transformed into BJ5183,which contained adenovirus backbone vector pAdEasy-1 to achieve the homologous recombination and obtain recombinant adenovirus genome plasmid named pAdeasy-N1. Then the pAdeasy-N1 was subsequently linearlized with pac I and transfected into HEK293 cells to form pAd-N1, pAdtrack-N1 and pAdeasy-N1 were identified by PCR, double-digestion and sequencing. GFP and RT-PCR were used to identify pAd-N1 whether the NA gene was expressed. The results showed that,pAdtrack-N1 and pAdeasy-N1 were correct,and the NA gene was expressed in HEK293 cells.
出处
《动物医学进展》
CSCD
北大核心
2010年第6期15-19,共5页
Progress In Veterinary Medicine
基金
河南省高校杰出科研人才创新工程项目(2006KYCX020)
关键词
腺病毒载体
AIV
H5N1亚型
NA基因
载体构建
recombinant adenovirus vector
Avian influenza virus H5N1
NA gene
vector construction
