鸭瘟病毒UL51基因在感染宿主细胞中的定位和转录特征  

Characterization of localization and transcription of duck plague virus UL51 gene in virus-infected host cells

作　　者：沈婵娟[1] 程安春[1,2] 汪铭书[1,2] 文明[1] 招丽婵[1] 贾仁勇[1] 徐超[1] 孙涛[1] 葛菡[1] 周登春[1] 余波[1] 陈孝跃[1,2] 

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]四川省动物疫病与人类健康重点实验室,四川雅安625014

出　　处：《中国兽医学报》2010年第9期1160-1166,共7页Chinese Journal of 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(PCSIRT08);教育部"新世纪优秀人才支持计划"项目(NCET-06-0818);教育部高等学校科技创新工程重大项目培育资金项目(706050);水禽现代农业产业技术体系岗位科学家项目(2008-2012);四川省基础研究重大项目(07JY029-016/07JY029-017);四川省重点建设学科项目(SZD0418)

摘　　要：对鸭瘟病毒(DPV)UL51基因在感染宿主细胞内的定位和转录时相进行分析,为进一步研究该基因的特性和功能提供有用的试验数据和材料。构建pET32a-UL51原核表达载体,将其转化至E.coliBL21(DE3)中进行IPTG诱导表达后,用Ni2+-NTA琼脂糖凝胶柱层析法纯化该表达产物。以纯化的重组蛋白为抗原免疫兔子,制备兔抗UL51多克隆抗血清,通过间接免疫荧光和RT-PCR法检测该基因在DPV感染的鸭胚成纤维细胞(DEF)内的定位和转录情况。间接免疫荧光结果显示,最早可在感染后8h的胞浆中检测到特异性荧光点,24～36h有大量绿色荧光团块聚集于近核区域,之后随着细胞病变的增强,胞浆中的绿色荧光开始减弱,且在感染晚期的胞核中也出现了少量弥散的绿色荧光。RT-PCR结果显示,DPVU L51基因从感染后4h开始转录,其后转录量不断增大,从6～48h一直保持在较高水平,之后又降低。DPVU L51蛋白主要定位于感染的宿主细胞的胞浆中特别是近核区域,与其在α-疱疹病毒中的同源物的定位结果一致;并且与其他几种α-疱疹病毒的转录情况对比后,可认为该基因是一个晚期(γ类)基因。This study aims to analyze the intracellular localization and transcription kinesics of duck plague virus （DPV）UL51gene in virus-infected cells,so that providing valuable experimental data and materials for further stud-ying the characteristic and function of this gene.The UL51gene was cloned into the prokaryotic expression vector pET-32a（＋）to generate the recombinant plasmid pET32a-UL51,and then transformed into E.coli BL21（DE3） strain.IPTG was added to induce a recombinant 6-His-UL51fusion protein.The fusion protein was purified by Ni 2＋- NTA agarose gel column chromatography,and used to immunize rabbit to generate the UL51polyclonal antiserum. Indirect immunofluorescence and RT-PCR were used to test the transcription and localization characteristic of this gene in DPV-infected duck embryo fibroblasts（DEF）.The result of immunofluorescence staining showed that specific fluorescence appeared in cytoplasm as early as 8hpost infection（p.i.）,and then a great deal of specific fluores-cence concentrated in the juxtanuclear region from 24 to 36hp.i.,but there after the specific fluorescence became to weaken followed by the appearance of cytopathic effects;meanwhile,little fluorescence was dispersed in the nuclear. The transcription analysis of this gene indicated that the transcripts can be firstly detected at 4hp.i.,and then the accumulation reached a peak from 6to 48h;60 hlater,the transcripts descended significantly to a relatively low lev-el.The UL51 protein mainly located to the cytoplasm,and especially to the juxtanuclear region,in line with that of the homologous proteins in alphaherpesviruses;Furthermore,in contrast to the transcription kinesics of other alpha-herpesviruses,we suggested UL51should be classified as late（γclass）gene.

关 键 词：鸭瘟病毒 UL51基因 原核表达 间接免疫荧光 RT-PCR 

分 类 号：S852.65[农业科学—基础兽医学]