Objective To construct the recombinant adenovirus vector carrying Livinct gene and green fluorescent protein ( GFP ) with AdEasy - 1 system. Methods Livinct gene was amplified with PCR and cloned into the pAdTrack -CMV shuttle vector, and subsequently cotransduced into E. Coll BJ5183 cells with pAdEasy - 1 plasmid for homologous recombination. The linearized recombinant plasmid was subsequently transfected into HEK293 cells. The recombinant adenovirus pAd - GFP - Livinct was detected by PCR and restriction enzyme digestion analysis. Results The recombinant plasmid and the recombinant adenovirus vectors pAd - GFP - Livinct were identified by restriction enzyme digestion analysis. Recombinant adenovirus pAd - GFP - Livinct 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 - Livinct has been successfully constructed , which facilitates further research of Livinct function.%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.
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