鸭瘟病毒UL26.5基因的克隆和编码蛋白的亚细胞定位  

Cloning of duck plague virus UL26.5 gene and subcellular localization of its encoded protein in virus-infected host cells

作　　者：张瑶[1] 程安春[1,2] 汪铭书[1,2] 贾仁勇[2] 朱德康[1,2] 刘菲[2] 罗启慧[2] 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]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11期941-949,共9页Chinese Veterinary Science

基　　金：教育部"长江学者和创新团队发展计划"创新团队项目(IRT0848);教育部高等学校科技创新工程重大项目培育资金项目(706050);现代农业产业技术体系建设专项资金项目(nycytx-45-12);四川省重大项目培育资金项目(07ZZ028)

摘　　要：对鸭瘟病毒(DPV)UL26.5基因(GenBank登录号EF643564)的分子特征分析表明,其具有疱疹病毒支架蛋白的典型特征,含有6个保守的结构功能域和1个丝氨酸蛋白酶水解位点(M位点),且具有与其功能相关的磷酸化位点;编码的多肽链是一种膜外蛋白。DPV UL26.5与α-亚科禽类疱疹病毒属的进化关系最近。将UL26.5基因扩增产物亚克隆至pET-32a(+)原核表达载体,并转化至E.coliBL21(DE3)中,经IPTG诱导表达,获得一约59 ku的表达蛋白,与预期大小一致。将重组蛋白纯化后,制备兔抗血清,经间接ELISA检测,效价达1∶204 800。DPV UL26.5基因编码蛋白亚细胞定位检测结果显示,最早可在感染后10 h的细胞核中检测到该蛋白,12 h后细胞核中出现较多绿色荧光,48 h时细胞质中也出现少量荧光。试验结果为进一步开展DPV UL26.5基因功能的研究提供了重要数据和材料。From the constructed duck plague virus（DPV） CHv strain genomic library,an UL26.5 gene was defined（GenBank accession number EF643564）.Molecular characteristic analysis showed that DPV UL26.5 gene contained six conserved motifs and a hydrolysis site of serine protease（M site） which were conserved in major scaffolding protein of herpesvirus.The phosphorylation sites were predicted.DPV UL26.5 protein was located outside of membrane.Phylogenetic studies demonstrated that DPV UL26.5 gene was the closest with poultry alpha herpesvirus.The DPV UL26.5 gene was amplified,and subcloned into the prokaryotic expression vector pET-32a（＋）.Then the recombinant plasmid was transformed into Escherichia coli BL21（DE3） strain and expressed under IPTG induction.SDS-PAGE analysis showed that the expressed protein was about 59ku of molecular weight.The protein was purified and used to immunize rabbit preparing anti-serum production.The ELISA antibody titer was up to 1∶204800.Moreover,the subcellular localization detection was observed using immunofluorescence technique.The results showed that the encoded protein DPV UL26.5 appeared in nucleus as early as 10 hours post-infection and the fluorescence increase in more nuclei after at hour 12 post-infection.After 48h,the fluorescence appeared in both cytoplasm and nucleus.Above all,the results provided significant data and material for the study on function of DPV UL26.5 gene and the pathogeny of DPV.

关 键 词：鸭瘟病毒 UL26.5基因 分子特性 亚细胞定位 

分 类 号：S852.659.1[农业科学—基础兽医学]