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

作　　者：赵俊丽[1] 王东阳[1] 南天惠[1] 王丽娴[1] 廖晓玉[1] 夏海滨[1] 

机构地区：[1]陕西师范大学生命科学学院,陕西西安710062

出　　处：《陕西师范大学学报（自然科学版）》2011年第6期60-63,共4页Journal of Shaanxi Normal University：Natural Science Edition

基　　金：国家自然科学基金资助项目(3087299331070137);陕西师范大学研究生培养创新基金资助项目(2010CXS018);陕西师范大学大学生创新基金资助项目(091071822)

摘　　要：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.

关 键 词：基因治疗 腺病毒 病毒示踪 病毒标记 衣壳蛋白pⅨ 

分 类 号：Q78[生物学—分子生物学] Q943.2