Zheng Xianxian, He Jinsheng, Fu Yuanhui, Xu Shaohua, Xie Can, Shi Changxin, Zhang Mei, Wang Xiaobo, Hong Tao
Department of Immunology, Anhui Medical University, Hefei 230032, China.
Sheng Wu Gong Cheng Xue Bao. 2010 Aug;26(8):1108-15.
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 2000 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.
为了在体外研究辅助依赖型腺病毒载体（HDAd）的转基因表达效率，我们构建了一种编码增强型绿色荧光蛋白（EGFP）的HDAd，命名为HDAd/EGFP，进行了大规模制备和纯化，然后在荧光显微镜和电子显微镜下对纯化后的HDAd/EGFP进行鉴定。用分光光度计测定HDAd/EGFP的浓度后，在体外将HDAd/EGFP的转基因表达效率与编码EGFP的第一代腺病毒载体（FGAd/EGFP）进行比较。因此，我们分别用HDAd/EGFP和FGAd/EGFP以每细胞2000个病毒颗粒（vp）感染A549细胞，并通过流式细胞术分析EGFP的表达水平。结果，在早期感染的A549细胞中，HDAd/EGFP的EGFP荧光表达率和荧光强度高于FGAd/EGFP。HDAd能够在体外即时且高效地表达转基因，是一种潜在的疫苗载体。
