摘要
利用口蹄疫病毒(FMDV)2A蛋白具有自我裂解的功能,将其作为连接肽构建携带有H5N1亚型AIVHA和NA基因的重组腺病毒表达载体,进而为AIV基因工程疫苗的开发以及相关诊断试剂的开发提供依据。采用融合PCR的方法扩增出含有H5N1 AIV HA-2A-NA的基因,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-HA-2A-NA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-HA-2A-NA,将pAdeasyd-H5经PacI线性化后转染HEK293细胞株包装出含有HA-2A-NA基因的腺病毒pAd-HA-2A-NA。结果表明,构建的含有目的基因的腺病毒穿梭质粒pAdtrack-HA-2A-NA和含有目的基因的腺病毒质粒pAdeasy-HA-2A-NA经PCR、双酶切及核苷酸测序测定无误。线性化后的pAdeasy-HA-2A-NA转染HEK293细胞包装成功获得腺病毒pAd-HA-2A-NA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。本试验构建的含有AIV H5N1亚型HA-2A-NA基因的重组腺病毒表达载体,将为进一步研究开发基因工程疫苗提供病毒模型。
FMDV 2A peptide was introduced as a linker to construct a recombinant adenovirus co-expression vector contained HA and NA genes of avian influenza virus strain H5N1. This vector could be used to study the roles of HA and NA gene in AIV pathopoiesis and acquired HA and NA proteins. Using overlapping PCR to amplify HA-2A-N1, which was further cloned into shuttle plasmid pAdTraek-CMV. The shuttle plasmid was transformed into BJ5183 which had already contained adenovirus backbone vector pAdEasy-1 to obtain the homologous recombination and recombinant adenovirus genome plasmid named pAdeasy-HA-2A-NA. Then the pAdeasy- HA-2A-NA was subsequently linearlized with Pac I and transfected into HEK293 cells to form pAd-HA-2A-NA, pAdtraek-HA-2A-NA and pAdeasy-H5 were identified by PCR ,double-digestion and sequencing. GFP and RT-PCR were used to identify whether the-HA-2A- NA gene was expressed. The results showed that,pAdtrack-HA-2A-NA and pAdeasy-HA-2A-NA were correct, and the HA and NA gene was expressed in HEK293 cells. Through this study the pAdeasy-HA-2A-NA was constructed,which could be used as an excellent tool to product HA protein and form a model virus to research HA gene＇ s roles.
出处
《生物技术通报》
CAS
CSCD
北大核心
2010年第10期172-177,共6页
Biotechnology Bulletin
基金
河南省高校杰出科研人才创新工程项目(2006KYCX020)
关键词
腺病毒裁体
AIV
H5N1亚型
HA和NA基因
载体构建
Recombinant adenovirus vector Avian influenza virus strain H5N1 HA and NA gene Vector construction
