携带野生型PTEN基因的腺病毒载体的构建  

Construction of recombinant adenovirus containing wild-type PTEN

作　　者：于林君[1] 祝善俊[1] 周裔忠[1] 田颖[1] 王江[1] 祝之明[2] 

机构地区：[1]第三军医大学新桥医院心内科,重庆400037 [2]第三军医大学大坪医院高血压内科,重庆400042

出　　处：《免疫学杂志》2005年第3期240-243,共4页Immunological Journal

基　　金：国家自然科学基金资助项目(30370584)

摘　　要：目的构建携带野生型PTEN(Phosphatase and tensin homolog deleted on chromosome ten)基因的腺病毒载体,为研究PTEN功能和作用机制提供手段。方法将野生型PTEN基因克隆入含有绿色荧光蛋白(Green fluorescence protein,GFP)基因的pAdTrack-CMV质粒,在含有pAdEasy-1病毒骨架的BJ5183大肠杆菌内进行同源重组;重组子通过脂质体介导转染AD293细胞,并在AD293细胞内包装为具有感染能力的病毒颗粒;通过反复感染扩增病毒以达到感染靶细胞的适当滴度,通过GFP表达来监控腺病毒扩增;Westernblot检测靶细胞内PTEN蛋白的表达。结果感染腺病毒载体的AD293细胞表达GFP,随着时间逐渐增强,并且出现明显的细胞病变效应(Cytopathiceffect,CPE),经过3轮扩增,病毒达到合适的滴度。受腺病毒感染心肌细胞内PTEN蛋白表达明显增高。结论成功构建了携带PTEN基因的腺病毒载体。Objective To construct the recombinant adenovirus containing wild-type PTEN. Methods PTEN cDNA was cloned into shuttle plasmid pAdTrack-CMV, which containing green fluorescence protein (GFP) gene.The plasmid was linearized by Pme Ⅰ and transformed into PJ5183 E.coli , where the plasmid was recombined with pAdEasy-1 by homologous recombination. The recombinant was transfected into AD293 cells by Lipofectamine 2000 for packaging the adenovirus, and then the recombinant adenovirus was traced by monitoring GFP expression under fluorescence microscope. PTEN protein expression in the infected cardiomyocytes was detected by Western blotting. Re- sults GFP was expressed in recombinant adenovirus infected AD293 cells. Cytopathic effect (CPE) was found in GFP expression, which was exten- ding and brightening as time goes by. After 3 cycles of amplification, the titer of adenovirus containing wild-type PTEN was brought up to indicated level . PTEN protein expression in the cardiomyocytes increased dramatically. Conclusion With methods mentioned above, the PTEN recombinant adenovirus is constructed successfully.

关 键 词：PTEN 腺病毒 重组 载体 

分 类 号：R346[医药卫生—基础医学]