TK基因缺失对鸭肠炎病毒在鸡胚成纤维细胞中复制影响的评价  

Evaluation on the effect of duck enteritis virus replication in CEF influenced by the deletion of TK gene

作　　者：王玉龙[1] 李慧昕[2] 李冰[2] 胡永浩[1] 刘胜旺[1,2] 

机构地区：[1]甘肃农业大学动物医学学院,甘肃兰州730070 [2]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/禽传染病研究室,黑龙江哈尔滨150001

出　　处：《中国预防兽医学报》2015年第5期339-343,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：蛋鸡体系(CAR-41-K12);国家科技支撑计划课题(2015BAD12B03)

摘　　要：为鉴定鸭肠炎病毒(DEV)的非必需基因,本研究利用增强型绿色蛋白(EGFP)基因为报告基因,通过在转染的鸡胚成纤维细胞(CEF)中同源重组,将其插入DEV基因组的TK基因中,经筛选和纯化绿色荧光病毒蚀斑,获得表达EGFP的重组DEV,命名为r DEVTK-EGFP。对重组病毒与亲本病毒DEV Clone-03复制动力学比较结果显示,重组病毒滴度显著低于亲本病毒(p<0.01)。对重组病毒在复制过程中感染细胞能力和荧光表达时相检测结果表明,在感染后48 h^72 h表达荧光信号细胞明显增多,该结果与重组病毒复制动力学一致。将重组病毒在CEF中连续传代至20代,仍具有遗传稳定性。该研究结果表明,TK基因为DEV复制非必需基因,可以作为外源基因插入位点,用于禽用新型基因工程疫苗的研制。To identify the nonessential gene of duck enteritis virus （DEV）, the enhanced green fluorescence protein gene was used as the report gene to insert into the genomic DNA of DEV by the method of homologous recombination. The recombinant virus was generated and purified by selecting the green fluorescence plaque and designated as rDEVTK-EGFP. By comparing the replication kinetics between the recombinant virus and the parental virus DEV Clone-03, it was showed that the titers of the recombinant virus was decreased significantly than that of the parental virus （p〈0.01）. The infection capability and the expression phase of the fluorescence signal was detected by flow cytometry, the fluorescence signal increased significantly fi＇om 48 to 72 hours post infection, which was consistent to the result of the replication kinetics. The recombinant virus was stable after being passed in CEF for 20 times. It was suggested that TK gene was nonessential for the replication of DEV and could be used as the insertion site for the foreign gene, which was meaningful for the study of avian gene engineering vaccine.

关 键 词：鸭肠炎病毒 TK基因 重组病毒 病毒复制 

分 类 号：S852.65[农业科学—基础兽医学]