一种经miniSOG标记的人腺病毒的制备  

Preparation of Recombinant Human Adenoviruses Labeled with miniSOG

作　　者：邹小辉[1] 肖蓉[1,2] 郭小娟[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传染病预防控制国家重点实验室,北京100052 [2]中国医学科学院药物研究所,北京100050

出　　处：《病毒学报》2016年第1期32-38,共7页Chinese Journal of Virology

基　　金：国家自然科学基金项目(31400156);国家科技重大专项课题(2013ZX10004-101)

摘　　要：为了探索腺病毒感染细胞后病毒核心在胞内运输的过程,本研究构建了一株经miniSOG标记IX蛋白的人腺病毒。首先,我们通过重叠延伸PCR的方法合成了miniSOG基因,将其克隆至pcDNA3表达载体后转染293细胞,荧光显微镜下可观察到miniSOG蛋白的表达,经固定,封闭,DAB-H2O2染色,OsO4固定染色后,可见表达miniSOG蛋白的细胞呈深褐色。之后将miniSOG构建至腺病毒穿梭载体pShuttle的IX基因3’端,并通过与骨架质粒pAdeasy-1在E.coli BJ5183细菌内同源重组获得了重组腺病毒质粒pAd5-IXSOG。pAd5-IXSOG质粒经PacI酶切线性化后转染293细胞,拯救出重组腺病毒HAdV-5-IXSOG。经过8轮扩增,超速离心纯化后获得2.0ml浓度为6.0×1011vp/mL的重组腺病毒,电子显微镜下可见完整病毒颗粒。本研究经反向遗传学技术对腺病毒载体进行改造,成功制备了经miniSOG标记的重组腺病毒HAdV-5-IXSOG,该病毒可用于腺病毒感染细胞的实时追踪。We wished to study the intracellular transport of adenoviruses.We constructed a novel recombinant adenovirus in which the structural protein IX was labeled with a mini-singlet oxygen generator（miniSOG）.The miniSOG gene was synthesized by overlapping extension polymerase chain reaction（PCR）,cloned to the pcDNA3 vector,and expressed in 293 cells.Activation of miniSOG generated sufficient numbers of singlet oxygen molecules to catalyze polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by transmission electron microscopy（TEM）.To construct miniSOG-labelled recombinant adenoviruses,the miniSOG gene was subcloned downstream of the IX gene in a pShuttle plasmid.Adenoviral plasmid pAd5-IXSOG was generated by homologous recombination of the modified shuttle plasmid（pShuttle-IXSOG）with the backbone plasmid（pAdeasy-1）in the BJ5183 strain of Eschericia coli.Adenovirus HAdV-5-IXSOG was rescued by transfection of 293 cells with the linearized pAd5-IXSOG.After propagation,virions were purified using the CsCl ultracentrifugation method.Finally,HAdV-5-IXSOG in 2.0mL with a particle titer of 6×1011vp/mL was obtained.Morphology of HAdV-5-IXSOG was verified by TEM.Fusion of IX with the miniSOG gene was confirmed by PCR.In conclusion,miniSOG-labeled recombinant adenoviruses were constructed,which could be valuable tools for virus tracking by TEM.

关 键 词：腺病毒 IX蛋白 miniSOG 病毒拯救 

分 类 号：R373.9[医药卫生—病原生物学]