人DeltaNp73α基因重组腺病毒载体的构建及其鉴定  

Construction of recombinant adenovirus carrying human DeltaNp73α cDNA with Adeasy system

作　　者：胡义杰[1] 范士志[1] 蒋耀光[1] 何勇[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所全军胸外科中心,重庆400042

出　　处：《第三军医大学学报》2007年第9期759-762,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30600611);第三军医大学中青年基金(XG200540)~~

摘　　要：目的利用Adeasy腺病毒载体系统构建含人DeltaNp73α cDNA的重组腺病毒并在293细胞中扩增制备重组病毒。方法从pcDNA3-DeltaNp73α-HA中酶切出DeltaNp73α基因,并插入pAdTrack-CMV中构建成腺病毒穿梭质粒;pAdTrack-CMV-DeltaNp73α线性化后电穿孔法转化到含有腺病毒骨架质粒pAdeasy-1的E.coli.BJ5183菌株内同源重组。筛选正确的重组质粒,PacⅠ线性化后脂质体法转染293T细胞包装成重组病毒颗粒;并在293T细胞中反复扩增;增强离心法转染树突状细胞,Western blot检测目的蛋白的表达。结果经限制性内切酶酶切鉴定、PCR筛选、基因测序和GFP表达证实成功构建了携带DeltaNp73α cDNA的重组腺病毒载体并制备出高滴度的重组病毒;Western blot检测出预期相对分子质量为67×103的特异条带。结论成功构建了携带DeltaNp73α cDNA的重组病毒。Objective To construct recombinant adenovirus carrying human DeltaNp73α cDNA. Methods Human DeltaNp73α cDNA was cloned into adenovirus transfer vector pAdTrack-CMV. The obtained plasmid, pAdTrack-CMV-DeltaNp73α, was then linearized with Pme Ⅰ and transformed into E. coli. strain BJ5183 containing pAdeasy-1, the viral DNA plasmid, for homologous recombination. The adenoviral recombinant was then cleaved with Pac Ⅰ and transfected into 293T cells to produce viral particles. The virus was then transfected into dentritic cells, and the expression of DeltaNp73ot was proved by GFP expression and Western blotting. Results Recombinants were confirmed right by restriction enzyme analysis, PCR on viral lysate and expression of green fluorscence protein. A band of 67 x 103 was observed by Western blotting as expected. Conclusion The recombinant adenovirus carrying human DeltaNp73α cDNA is successfully constructed, paving the way for further research about its high expression in interested cells.

关 键 词：重组腺病毒 DehaNp73α 

分 类 号：R373[医药卫生—病原生物学] R394.33[医药卫生—基础医学]