表达绿色荧光蛋白的重组1型禽腺病毒的构建  

Construction of Recombinant Fowl Adenovirus Serotype 1 Exprsessing Exotic Green Fluorescence Protein

作　　者：周洁 赵莉[1,2,3] 胡建华 高诚 

机构地区：[1]上海实验动物研究中心,上海201203 [2]扬州大学兽医学院,扬州225009 [3]中科院上海药物所,上海201203

出　　处：《实验动物与比较医学》2013年第4期249-255,共7页Laboratory Animal and Comparative Medicine

基　　金：上海市科委基金项目（No.09140901400）

摘　　要：目的构建表达绿色荧光蛋白的重组禽腺病毒。方法通过带有禽腺病毒基因组复制非必需区（nt40065～nt43684）侧翼序列和增强型绿色荧光蛋白基因的转移质粒与亲本禽腺病毒在鸡肝癌细胞内同源重组，缺失掉了两侧翼序列之间的复制非必需区而替换为增强型绿色荧光蛋白基因的完整表达盒。为提高同源重组率，对质粒转染细胞的条件进行了摸索，确定了最佳转染条件为细胞以3×10^5／ml密度接种到6孔板上，36h后用8μl lipofectamine 2000试剂和3她pUC—LR—eGFP质粒转染，蚀斑方法筛选重组病毒。结果获得了表达EGFP的重组病毒单一克隆株rFAV—I-eGFP。结论研究结果为开展禽类活载体疫苗研制及相关基础研究提供了有利参考。Objective To construct a recombinant fowl adenovirus expressing exotic green fluores- cence protein（eGFP）. Methods The flanking fragments of non-essential region（nt40 065-nt43 684） in fowl adenovirus genome were cloned into a transfer plasmid as well as eGFP. Through homologous recombination between the transfer plasmid and wild fowl adenovirus in chicken hepatocellular carci- noma cell line（LMH）, the non-essential region between the flanking fragments was replaced by the complete eGFP expression cassette. In order to improve the occurrence probability of recombination, the transfection conditions such as logarithmic growth phase of LMH, the ratio of plasmid and lipofectamine, cells seeding density were explored. The optimum transfection condition was determined as follows： Seed 3×10^5/ml LMH cells into 6 cell culture plate, 36 h later, cells were transfected with 8 μl lipofectamine 2 000 and 3.0 μg plasmid pUC-LR-eGFP. Recombinant virus was screening by plaque purification. Results recombinant adenovirus serotype 1 （rFAdV- 1） expressing eGFP was obtained. Conclusion These results provided favorable references for fowl live vaccine development and related fundamental research.

关 键 词：重组I型禽腺病毒 鸡肝癌细胞系 增强型绿色荧光蛋白 

分 类 号：S852.65[农业科学—基础兽医学]