GFP标记Ⅸ蛋白的重组腺病毒的构建与鉴定  

Construction and identification of recombinant adenovirus with GFP labeled protein Ⅸ

作　　者：邹小辉[1] 郭小娟[1] 王敏[1] 屈建国[1] 鲁茁壮[1] 洪涛[1] 

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京100052

出　　处：《中华实验和临床病毒学杂志》2016年第2期204-207,共4页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金（31400156）

摘　　要：目的 为了更加直观地观察腺病毒（Adenovirus,Ad）与细胞之间的相互作用,本研究拟构建一株经绿色荧光蛋白（Green Fluorescent Protein,GFP）标记（pⅨ）蛋白的5型重组腺病毒（Ad5-IXGFP）.方法 我们将GFP基因构建至腺病毒穿梭质粒pShuttle中Ⅸ基因3＇端,通过与骨架质粒pAdeasy-1在E.coli BJ5183细菌内的同源重组,获得了重组腺病毒质粒pAd5-IXGFP.将pAd5-IXGFP质粒经Pac Ⅰ酶切线性化后转染293细胞,拯救出重组腺病毒Ad5-IXGFP.结果 经扩增和超速离心纯化后可获得浓度为6.9＆#215;1011 vp/ml的重组腺病毒.该病毒于4℃与HEp-2细胞孵育后,重组病毒吸附于HEp-2细胞表面,用荧光显微镜观察,可见绿色荧光.结论 利用反向遗传学技术对重组腺病毒外壳蛋白pⅨ进行修饰,成功制备的经GFP标记的重组腺病毒可用于实时动态追踪腺病毒颗粒感染细胞的过程.Objective To study the role of protein Ⅸ（pⅨ） in the interaction between adenovirus and host cell dynamically,we attempt to construct a recombinant human adenovirus with the capsid protein pⅨ being labelled with Green Fluorescent Protein （GFP）.Methods GFP gene was inserted into the downstream of pⅨ genein the shuttle plasmid to form pShuttle-IXGFP;and adenoviral plasmid pAd5-IXGFP was generated by homologous recombination of pShuttle-IXGFP with the backbone plasmid （pAdeasy-1） in E.coli BJ5183 strain.Recombinant adenovirus （Ad5-IXGFP） was rescued by transfecting 293 cells with the linearized pAd5-IXGFP.Results After 4 rounds of propagation in 293 cells,viruses were purified with CsCl ultracentrifugation method.Ad5-IXGFP in 1.0 ml with a particle titer of 6.9 ＆#215; 1011 vp/ml was finally obtained.PCR was performed to confirm the fusion of Ⅸ with GFP gene in the genome of Ad5-IXGFP.Virions were incubated with suspended HEp-2 cells at 4 C for 5 h,and the attachment of Ad5-IXGFP on the cellular membrane could be observed under fluorescence microscope.Conclusion GFP-labelled recombinant adenovirus was successfully constructed,which could be a valuable tool for subcellular localization fo pⅨ after virus entry real-time single adenovirus tracking by confocal fluorescence microscopy.

关 键 词：腺病毒 衣壳蛋白IX 绿色荧光蛋白 荧光标记 

分 类 号：R373[医药卫生—病原生物学]