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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。
The construction of helper-dependent adenoviral vector (HDAd), which can express enhanced green fluorescent protein (EGFP), was completed, and a lot of preparation, purification and identification of in vitro expression were completed. Fluorescence microscopy confirmed HDAd / EGFP expression, electron microscopy observed CsCl purified adenovirus typical morphology. The virus concentration was determined to be 4.0 × 1012 Virus particles (vp) / mL by spectrophotometer. In vitro infection and transgene expression efficiency comparison with EGFP-expressing first generation adenoviral vector (FGAd) FGAd / EGFP A549 cells were infected with about 2000 vp / cell of HDAd / EGFP and FGAd / EGFP, respectively Cell, flow cytometry detection of EGFP expression. By analyzing the expression of EGFP by flow cytometry at the same time point, we found that HDAd / EGFP had higher fluorescence and higher expression intensity than that of FGAd / EGFP in early stage of HDAd / EGFP infection, indicating that HDAd vector has transiently high transgene expression Characteristics, is a more valuable vaccine vector.