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

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2 000 vp/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。

Abstract:

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 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.