辅助病毒依赖型腺病毒载体转基因的体外表达效率  

In vitro transgenic expression efficacy of a helper-dependent adenoviral vector encoding enhanced green fluorescent protein

作　　者：郑娴娴[1,2] 何金生[1,2] 付远辉[2] 徐少华[2] 谢灿[1] 石长信 张梅[1] 王小波[1,2] 洪涛[2,4] 

机构地区：[1]安徽医科大学免疫学教研室,合肥230032 [2]北京交通大学生命科学与生物工程研究院,北京100044 [3]Division of Hernatology-Oncology,Mayo Clinic,Scottsdale AZ 85259,USA [4]中国疾病预防控制中心病毒病蓣防控制所,北京100052

出　　处：《生物工程学报》2010年第8期1108-1115,共8页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(No.30671965)资助~~

摘　　要：构建可表达增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)的辅助病毒依赖型腺病毒载体(Helper-dependent adenoviral vector,HDAd),并完成大量制备、纯化和体外表达鉴定。荧光显微镜证实HDAd/EGFP可表达,电镜下观察到经CsCl纯化后的腺病毒的典型形态。分光光度计法测定病毒的浓度为4.0×1012颗粒数(Virus particle,vp)/mL。与可表达EGFP的第一代腺病毒载体(First generation adenoviral vector,FGAd)FGAd/EGFP进行了体外感染和转基因表达效率的比较研究,分别用约2000vp/细胞的HDAd/EGFP和FGAd/EGFP感染A549细胞,流式细胞仪检测EGFP的表达情况。通过相同时间点流式细胞仪分析EGFP的表达情况,可见HDAd/EGFP感染早期的A549细胞较FGAd/EGFP有更高的荧光表达率及更高的表达强度,显示HDAd载体具有转基因瞬时高表达的特性,是一种更有价值的疫苗载体。To investigate the transgenic expressing efficacy of helper-dependent adenoviral vector （HDAd） in vitro, we constructed a HDAd encoding enhanced green fluorescent protein （EGFP）, denominated as HDAd/EGFP, performed large scale preparation and purification, and then identified the purified HDAd/EGFP under fluorescent microscope and electron microscope. After the concentration of HDAd/EGFP was determined by spectrophotometer, the transgenic expression efficiency of HDAd/EGFP was compared with first generation adenoviral vector encoding EGFP （FGAd/EGFP） in vitro. Therefore, we infected A549 cells with 2 000 virus particles （vp） per cell by HDAd/EGFP and FGAd/EGFP respectively and analyzed EGFP expressing level by flow cytometry. Consequently, the fluorescent expression rate and fluorescent intensity of EGFP were higher in early infected A549 cells by HDAd/EGFP than by FGAd/EGFP. HDAd, capable of expressing transgene instantly and efficiently in vitro, is a potential vaccine vector.

关 键 词：辅助病毒依赖型腺病毒载体 第一代腺病毒载体 增强型绿色荧光蛋白 转基因表达 

分 类 号：R373[医药卫生—病原生物学]