鸭瘟病毒UL51基因在COS-7细胞中的瞬时表达  

Transient expression of duck plague virus UL51 gene in COS-7 cells

作　　者：沈婵娟[1] 程安春[1,2] 汪铭书[1,2] 郭宇飞[1,2] 信洪一[1] 徐超[1] 贾仁勇[1] 韩新峰[1] 

机构地区：[1]四川农业大学动物医学院禽病防治研究中心,四川雅安625014 [2]动物疫病与人类健康四川省重点实验室,四川雅安625014

出　　处：《中国兽医科学》2009年第10期859-865,共7页Chinese Veterinary Science

基　　金：国家自然科学基金项目(30771598);教育部"长江学者和创新团队发展计划"创新团队项目(IRT0848);现代农业产业技术体系建设专项项目(nycytx-45-12)

摘　　要：为阐明鸭瘟病毒(DPV)UL51基因的特性和功能,根据DPVUL51基因序列设计了1对特异性引物,用PCR方法扩增UL51基因,并将其克隆至pMD18-T载体上,经双酶切和测序鉴定后,再将该目的片段亚克隆到pcDNA3.1(+)真核表达载体上,得到重组质粒pcDNA3.1-UL51,通过脂质体介导将其转入COS-7细胞;应用实时荧光RT-PCR、Western-blotting和间接免疫荧光法检测UL51基因在COS-7细胞中的转录、表达和定位情况。结果表明,UL51基因在转染后6 h即已开始转录,12 h开始表达,24 h时转录和表达量均达最高峰,之后逐渐降低。该基因在COS-7细胞中表达蛋白的分子质量为33 ku,比预测的分子质量(约27.1 ku)大。间接免疫荧光法显示,该基因产物早期聚集于COS-7细胞的近核区域,晚期位于胞浆和胞核中。To elucidate the characteristics and functions of duck plague virus（DPV） ULS1 gene,a pair of primers was designed based on the sequence of DPV ULS1 gene, and the gene was amplified by PCR, then cloned into pMD18-T vector. After restriction enzyme digestion and sequence analysis, it was sub- cloned into the eukaryotic expression vector pcDNA3. 1 to generate the recombinant plasmid pcDNA3. 1- UL51,which was transfected into the COS-7 cells by Lipofectin transfection. Then, its transcription,expression and localization were determined by real-time RT PCR,Western-blotting and indirect immunofluorescence assay. The results indicated that its transcripts appeared at hour 6 expression level post-transfection（PT） ,and its expression product was first detected at hour 12 PT,and then the expression product was up to a peak at hour 24 PT,thereafter the expression level was reduced. A 33 ku protein in COS-7 cells was detected,which was larger than the predicted 27.1 ku. The UL51 protein was localized to the perinuclear regions at hour 12 PT,and to the nucleus and cytoplasm at later time.

关 键 词：鸭瘟病毒 UL51基因 真核表达载体 COS-7细胞 实时荧光RT-PCR 免疫印迹 间接免疫荧光法 

分 类 号：S852.659.1[农业科学—基础兽医学] Q786[农业科学—兽医学]