Preliminary study of the UL55 gene based on infectious Chinese virulent duck enteritis virus bacterial artificial chromosome clone.
Preliminary study of the UL55 gene based on infectious Chinese virulent duck enteritis virus bacterial artificial chromosome clone.
复制标题
基于传染性中华强毒鸭肠炎病毒细菌人工染色体克隆的UL55基因初步研究
DOI：
10.1186/s12985-017-0748-y
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发表时间：
2017-04-13
期刊：
影响因子：
4.8
通讯作者：
Cheng A
中科院分区：
文献类型：
--
作者：
Wu Y;Li Y;Wang M;Sun K;Jia R;Chen S;Zhu D;Liu M;Yang Q;Zhao X;Chen X;Cheng A
鸭致死性肠炎病毒(DEV)感染可引起禽形目多种水禽的高发病率和高死亡率。然而，由于DEV基因组在宿主细胞中的维持和操作的障碍，包括保守的UL55基因在内的病毒基因在宿主细胞中的功能知之甚少。方法采用同源重组的方法，将包含BAC mini-F序列和选择标记EGFP的转移载体插入到UL23基因中，构建了鸭肠炎病毒中国强毒株(DEV CHV)的感染性细菌人工染色体(BAC)克隆。用两步红色重组法产生UL55缺失及其回复突变体。拯救的重组病毒的克隆基础。通过生长特性和共定位分析，研究了UL55基因在DEV复制中的作用及其对UL26.5蛋白分布的影响。结果DEV CHV全基因组可在大肠杆菌中以BAC克隆形式稳定保存，并在DEF细胞中进行重组。以新城疫病毒CHV-BAC-G为基础构建的UL55缺失突变体在感染鸭胚胎成纤维细胞(DEF)中的生长曲线、空斑形态和病毒滴度与亲本病毒相似。免疫荧光检测表明，UL55基因缺失不影响UL26.5蛋白在细胞内的分布。这些数据也提示，DEV CHV的感染性BAC克隆将有助于DEV基因组的基因功能研究。结论我们首次成功地获得了临床致死株DEV CHV的感染性BAC克隆。基于该平台构建的UL55基因突变体将为DEV基因的功能研究提供一个非常有用的工具。我们发现，最不为人所知的DEV UL55对病毒复制和UL26.5分布是必不可少的，它可能是开发双价疫苗的一个非常有前途的候选基因。目前正在进行实验，以验证UL55基因位点作为外源基因插入位点用于开发DEV载体疫苗的可能性。
BackgroundLethal Duck Enteritis Virus (DEV) infection can cause high morbidity and mortality of many species of waterfowl within the order Anseriformes. However, little is known about the function of viral genes including the conserved UL55 gene among alpha herpes virus due to the obstacles in maintenance and manipulation of DEV genome in host cells.MethodsIn this paper, we constructed an infectious bacteria artificial chromosome (BAC) clone of the lethal clinical isolate duck enteritis virus Chinese virulent strain (DEV CHv) by inserting a transfer vector containing BAC mini-F sequence and selection marker EGFP into UL23 gene using homologous recombination. UL55 deletion and its revertant mutant were generated by two-step RED recombination inE. colion basis of rescued recombinant virus. The function of UL55 gene in DEV replication and its effect on distribution of UL26.5 protein were carried out by growth characteristics and co-localization analysis.ResultsThe complete genome of DEV CHv can be stably maintained in E. coli as a BAC clone and reconstituted again in DEF cells. The generated UL55 deletion mutant based on DEV CHv-BAC-G displayed similar growth curves, plaque morphology and virus titer of its parental virus in infected Duck Embryo Fibroblast (DEF) cells. Immunofluorescence assay indicated that the loss of UL55 gene do not affect the distribution of UL26.5 protein in intracellular. These data also suggest infectious BAC clone of DEV CHv will facilitate the gene function studies of DEV genome.ConclusionsWe have successfully developed an infectious BAC clone of lethal clinical isolate DEV CHv for the first time. The generated UL55 gene mutant based on that demonstrated this platform would be a very useful tool for functional study of DEV genes. We found the least known DEV UL55 is dispensable for virus replication and UL26.5 distribution, and it could be a very promise candidate locus for developing bivalent vaccine. Experiment are now in progress for testifying the possibility of UL55 gene locus as an exogenous gene insertion site for developing DEV vectored vaccine.