表达绿色荧光蛋白突变体的重组伪狂犬病毒的构建及其增殖特性  

Construction and Growth Properties of Recombinant Pseudorabies Virus Expressing Modified Enhanced Green Fluorescent Protein

作　　者：方六荣[1] 陈焕春[1] 肖少波[1] 张辉[1] 牛传双[1] 

机构地区：[1]华中农业大学畜牧兽医学院动物病毒研究室,武汉430070

出　　处：《生物化学与生物物理学报》2002年第6期757-762,共6页

基　　金：国家自然科学基金资助项目 (No .3 9970559)~~

摘　　要：将绿色荧光蛋白突变体M 1(EGFPS14 7/P)基因融合到伪狂犬病毒 (PRV)非必需糖蛋白 gG的第 8个氨基酸下游 ,通过同源重组、空斑纯化和PCR筛选获得能表达M 1并导致gG基因部分缺失的重组病毒 gG-/M1+ 。重组病毒经Southern杂交、Western印迹和荧光观察证实构建正确。纯化的重组病毒以低感染指数接种PK 15细胞 ,在感染早期 (6h)就能观察到荧光 ,随着病毒的增殖 ,荧光逐渐增强 (2 4～ 36h) ,直至完全病变 ,荧光淬灭。进一步对重组病毒gG-/M1+ 与亲本株gG-/LacZ+ 、野毒株的增殖特性进行比较 ,发现 3种毒株在增殖滴度上无显著差异。上述结果表明构建的PRVgG-/M1+ 突变株能作为活细胞示踪实时监测病毒感染的动态分析。The 1.0 kb DNA fragment containing the modified enhanced green fluorescent protein CDNAM1 (EGFP S147/P) andSV40 poly(A) signal sequence was amplified and cloned in frame behind the first eight codons of the nonessential glycoprotein G (gG) of pseudorabies virus (PRV) to yield a transfer plasmid. The transfer plasmid was linearized and cotransfected with the genomic DNA of PRV Ea mutant gG-/LacZ+. The resulting recombinant virus expressing M1, designated as gG-/M1+ , was isolated and confirmed by plaque purification, PCR, Southern blot and Western blot. PK-15 cells were infected with the purified recombinant virus at 0.1 pfu/cell and fluorescence emission was monitored at different times post-infection (p.i.) using fluorescence microscopy. Fluorescence emission could be detected as early as 6 h p.i. when there was no apparent cytopathic effects (CPE) yet. Fluorescence intensity increased drastically later. Maximum intensity was achieved at 24—36 h p.i. and fluorescence was stable. With the progress of the CPE, fluorescence vanished. The growth properties of gG-/M1+ in tissue cultures were further examined and the titer of gG-/M1+ was similar to that of PRV Ea wild strain and the parental strain gG-/LacZ+. The above results indicated that the recombinant virus expressing the modified EGFP can be used as anin vivo marker to monitor replication and spread, as well as to study the molecular pathogenesis of PRV.

关 键 词：表达 绿色荧光蛋白突变体 重组伪狂犬病毒 构建 增殖特性 标记 

分 类 号：Q785[生物学—分子生物学]