人AWP1重组腺病毒的构建及其在人血管内皮细胞中的表达和定位  

Construction of Human Flag-AWP1 Recombinant Adenovirus Vector and Its Expression and Localization in Human Vascular Endothelial Cell

作　　者：曹永宽[1] 莫永炎[2] 刘志峰[2] 薛刚[1] 王培红[1] 姜勇[2] 田伏洲[1] 

机构地区：[1]成都军区总医院全军普通外科中心,成都610083 [2]南方医科大学病生教研室,广州510515

出　　处：《中国普外基础与临床杂志》2009年第12期987-990,共4页Chinese Journal of Bases and Clinics In General Surgery

摘　　要：目的构建人flag-AWP1(associated with protein kinase C related kinase1)基因的重组腺病毒载体作为AWP1的转基因工具,研究AWP1在人血管内皮细胞ECV304中的表达与定位。方法将flag-AWP1基因亚克隆到腺病毒穿梭载体pAdTrack-CMV,经酶切鉴定正确后PmeⅠ线性化,转化至带有骨架质粒pAdEasy-1的大肠杆菌BJ5183中进行重组;经PacⅠ酶切鉴定和PCR鉴定、线性化后,用Lipofectamine转染293细胞进行包装、反复感染以获取高滴度重组病毒上清。再用获得的重组腺病毒Ad-flag-AWP1感染ECV304细胞,激光扫描共焦显微镜下观察AWP1在ECV304细胞中的表达与定位。结果包装病毒颗粒的PCR分析显示,成功获得了重组腺病毒Ad-flag-AWP1,该重组腺病毒能够高效感染ECV304细胞,并在激光扫描共焦显微镜下观察到AWP1在细胞内成功表达,且定位于细胞膜内侧。结论成功构建了flag-AWP1的重组腺病毒基因转染载体,AWP1在ECV304细胞中的表达与定位提示AWP1可能具有细胞信号转导因子的潜能,尚需进一步研究。Objective To construct AWP1 （associated with protein kinase C related kinase 1） recombinant adenovirus as the tool of transferring the gene and investigate its expression and localization in human vascular endothelial cell ECV304.Methods Cloned AWP1 cDNA was inserted into the multiply clone sites （MCS） of plasmid pcDNA3 for adding flag tag,and the flag-AWP1 gene was subcloned into shuttle vector pAdTrack-CMV.After identified with restrictional enzymes,plasmid pAdTrack-flag-AWP1 was linearized by digestion with restriction endonuclease Pine I , and subsequently cotransformed into E. coli BJ5183 cells with adenoviraI backbone plasrnid pAdEasy-1 to make homologous recombination. After linearized by Pac I , the homologous recombinant adenovirus plasmid transfected into 293 cells with Lipofectamine to pack recombinant adenovirus. After PCR assay of recombinant adenovirus granules, recombinant adenoviruses infected 293 cells repeatedly for obtaining the high level adeno- viruses solution. And then, the recombinant adenoviruses infected human ECV304 cells for observing the expression and localization of AWP1 under laser scanning confocal microscope （LSCM）. Results PCR assay showed that re combinant adenovirus Ad-flag-AWPl was obtained successfully~ and ECV304 cells were infected high-efficiently by the homologous recombinant virus. Then, it was observed that flag-AWP1 protein expressed in ECV304 cells and distributed in the leading edges of the cell membrane. Conclusion The vectors of flag-AWPl recombinant adenovirus are constructed, and the localization of AWP1 protein in ECV304 cells might show that AWP1 may be a potential role on the cell signal transduction.

关 键 词：AWP1 重组腺病毒 ECV304细胞 载体构建 细胞信号转导 

分 类 号：R373[医药卫生—病原生物学]