携带凋亡抑制蛋白Livinα基因的重组腺病毒载体的构建  

Construction of recombinant Livinα carrying adenovirus vector

作　　者：谢楚海[1] 吴波以[1] 付霞霏[2] 胡海澜[1] 

机构地区：[1]广州医学院第二附属医院骨科,广州510260 [2]南方医科大学珠江医院妇产科,广州510282

出　　处：《广东医学》2012年第5期583-586,共4页Guangdong Medical Journal

基　　金：广东省自然科学基金博士科研启动项目(编号:10451018201004365);广州医学院博士启动/留学归国启动基金资助项目(编号:2008C21)

摘　　要：目的构建携带凋亡抑制蛋白Livinα和绿色荧光蛋白(GFP)基因的重组腺病毒pAd-Livinα。方法 PCR扩增Livinα基因片段,克隆至穿梭质粒pAdTrack-CMV中,再与骨架质粒pAdEasy-1在大肠杆菌BJ5183中同源重组,在人胚肾293细胞中包装扩增得到携带Livinα和GFP基因的重组腺病毒pAd-GFP-Livinα。采用PCR对重组腺病毒进行鉴定。结果经酶切及PCR鉴定证实携带Livinα的重组腺病毒载体构建成功,并可在人胚肾293细胞中表达,病毒滴度2.15×1010 pfu/mL。结论成功构建了携带Livinα和GFP基因的重组腺病毒载体系统,为进一步研究Livinα的生物学特性奠定了基础。Objective To construct the recombinant adenovirus vector carrying Livinα gene and green fluorescent protein（GFP） with AdEasy-1 system.Methods Livinα gene was amplified with PCR and cloned into the pAdTrack-CMV shuttle vector,and subsequently cotransduced into E.coli BJ5183 cells with pAdEasy-1 plasmid for homologous recombination.The linearized recombinant plasmid was subsequently transfected into HEK293 cells.The recombinant adenovirus pAd-GFP-Livinα was detected by PCR and restriction enzyme digestion analysis.Results The recombinant plasmid and the recombinant adenovirus vectors pAd-GFP-Livinα were identified by restriction enzyme digestion analysis.Recombinant adenovirus pAd-GFP-Livinα was confirmed by PCR and successfully expressed in HEK293 cells with virus titer of 2.15×1010 pfu/mL.Conclusion The recombinant virus pAd-GFP-Livinα has been successfully constructed,which facilitates further research of Livinα function.

关 键 词：Livinα 腺病毒 绿色荧光蛋白 

分 类 号：R394[医药卫生—医学遗传学]