摘要
试验利用Gateway技术构建含山羊SKIP基因的重组腺病毒载体。首先采用PCR扩增山羊SKIP基因连接入pcDNA3.1(+),将酶切回收的SKIP产物片段克隆至腺病毒穿梭载体pYr-adshuttle-2获得重组质粒pYr-adshuttle-2-SKIP。从该重组质粒上,利用LR同源重组将SKIP-EGPF表达框转移至腺病毒骨架质粒pAd/PL-DEST,获得重组腺病毒质粒pAd-SKIP。该质粒经pacI线性化后转染HEK293A细胞包装病毒,获得重组腺病毒rAd-SKIP。重组腺病毒分别经酶切和PCR等方法进行鉴定。荧光显微镜观察转染效果。结果证实腺病毒载体中含有SKIP基因的目的片段,荧光显微镜发现该重组腺病毒能在C2C12细胞中高效表达。表明成功构建了同时表达SKIP蛋白和绿色荧光蛋白的重组腺病毒,为下一步开展关于SKIP基因的功能研究奠定了基础。
In this study, the Gateway technology was used to construct a recombinant adenovirus vector pAd-SKIP. The SKIP gene was amplified first by PCR and inserted into pcDNA3.1（＋） vector, then cloned into adenovirus adshuttle vector pYr-ad- shuttle-2 to obtain recombinant plasmid pYradshuttle-2-SKIP. The expression cassette of SKIP-EGPF was transferred to pAd / PL-DEST with LR recombinant reaction to acquire recombinant adenovirus plasmid pAd-SKIP-EGFP. The correct clone was linearized and tranformed into 293A cells. Recombinant adenovirus pAd-SKIP was obtained and identified. The transfection efficiency was observed under the fluorescent microscope. The results confirmed that the recombinant adenovirus vector con- tained goat SKIP gene. This vector was found to infect C2 C12 cells efficiently. It indicated that the recombinant adenovirus ex- pressing SKIP gene and green fluorescence was successfully constructed, laying the foundation for further study.
出处
《湖北农业科学》
北大核心
2013年第21期5258-5261,共4页
Hubei Agricultural Sciences
基金
湖北省农业科学院青年科学基金项目(2011NKYJJ16)
湖北省农业科技创新中心资助项目(2011-620-001-003)
