红色荧光蛋白遗传标记腺病毒衣壳蛋白pⅨ的实验研究

Study on capsid protein p Ⅸ genetically labeled by red fluorescent protein

采用在细菌Bj5183中同源重组的方法成功构建了红色荧光蛋白mCherry标记pⅨ蛋白的腺病毒质粒载体.将PacⅠ线性化的腺病毒质粒载体转染HEK293细胞7～10d后获得病毒裂解液,然后将其在体外进一步扩增并经CsCl密度梯度离心纯化后获得mCherry标记腺病毒pⅨ蛋白的病毒颗粒.在采用荧光显微镜检测荧光标记的病毒颗粒之后,将其加入A549细胞中,并在不同时间点追踪观察荧光标记的病毒颗粒在细胞内的定位.结果表明,在荧光显微镜下观察到病毒颗粒的红色荧光;在病毒与细胞孵育过程中,随着荧光标记病毒与A549细胞孵育时间的延长,病毒颗粒逐渐向细胞核周围聚集.

Adenoviral DNA vector genetically modified with pⅨ-mCherry was successfully constructed by means of homologous recombination in E.coli.Bj5183.The virus lysates were obtained after the transfection of adenovirus plasmid linearized by Pac Ⅰ into HEK293 cells for 7~10 days.Adenoviral lysates were propagated,and then adenoviral particles labeled with pⅨ-mCherry were purified by CsCl density gradient centrifugation.Purified virus particles were examined under fluorescence microscope,and the localization of fluorescently labeled adenovirus was then investigated by applying purified viruses into A549 cells at different time points.The results indicated that adenovirus particles were found to show red fluorescence under fluorescence microscope.With the prolonged incubation time of fluorescently labeled adenovirus with A549 cells,the viral particles were gradually localized around nucleus.