重组人p66Shc／腺病毒表达载体的构建与表达  

Construction and Expression of Hmnan Recombinant p66Shc Adenovirus

作　　者：徐蓉[1] 胡刚[2] 林雅军[2] 柳江 魏洁[2] 程建华[2] 许晓东[4] 孙洪范[1] 

机构地区：[1]北京协和医学院中国医学科学院生物医学工程所天津市生物医学材料重点实验室,天津市300192 [2]卫生部北京医院/卫生部北京老年医学研究所卫生部老年医学重点实验室,北京市100730 [3]江西省南昌市第三医院内分泌代谢科,南昌市330009 [4]卫生部北京医院血液科,北京市100730

出　　处：《医学分子生物学杂志》2012年第5期320-325,共6页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.81001439）,天津科技计划应用基础与前沿计划重点项目（No.08JCZDJC16000）

摘　　要：目的构建人p66Shc／腺病毒表达载体，观察其在内皮细胞中的表达。方法扩增p66Shc基因并与腺病毒骨架载体进行体外重组连接，经筛选、测序确认后转染进HEK293A细胞进行包装，10d后收集细胞裂解液感染HEK293A细胞和脐静脉内皮细胞，48h后分别用Western印迹和免疫荧光细胞化学法检测p66Shc蛋白表达水平。结果经测序确认目的基因p66Shc成功克隆入腺病毒载体中，转染HEK293A细胞后可在显微镜下观察到特征性的细胞病变效应，Western印迹检测和免疫荧光细胞化学法检测均表明感染了AdenoX-p66的HEK293A细胞和内皮细胞中p66Shc蛋白表达水平显著升高。结论人p66Shc／腺病毒表达载体构建成功并能在内皮细胞中表达。Objective To construct the human recombinant p66Shc adenovirus and observe the expression in human umbilical vein endothelial cells （HUVECs） . Methods Human p66Shc gene was amplified by PCR from pcDNA3.1 his-p66Shc and then directly cloned into the linearized adeno- viral vector in vitro. After confirmed by screening and gene sequencing, the linearized recombinant plasmid was transfeeted into HEK293A cells for packaging. The p66Shc expression in HEK293A cells and HUVECs infected by Adeno X-p66She was detected by Western Blot and Immunofluores- cence cytochemistry staining techniques. Results Gene sequencing indicated that pAdeno X- p66Shc contained p66Shc gene. The cytopathic effect was observed in the HEK293A cells at 10 days after pAdeno X-p66Shc transfection. The expression of p66Shc protein was significantly upregulated in HEK293A cells and HUVECs at 48h after AdenoX-p66Shc infection. Conclusion The Adeno- X Adenoviral System3 （CMV） can be used to efficiently and conveniently construct recombinant adenovirus containing p66Shc gene capable of efficiently infecting HUVECs.

关 键 词：P66SHC 腺病毒 同源重组 脐静脉内皮细胞 

分 类 号：Q782[生物学—分子生物学]