潜伏感染期伪狂犬病病毒DNA甲基化图谱测定及功能解析

项目名称： 潜伏感染期伪狂犬病病毒DNA甲基化图谱测定及功能解析

项目编号： No.31272567

项目类型： 面上项目

立项/批准年度： 2013

项目学科： 农业科学

项目作者： 陈陆

作者单位： 河南农业大学

项目金额： 77万元

中文摘要： 本项目拟通过免疫共沉淀富集甲基化DNA片段结合芯片分析测定伪狂犬病病毒(PRV)潜伏感染期基因组甲基化图谱，并应用甲基特异性的PCR扩增技术，对潜伏感染期和急性感染期与PRV基因组复制与转录起始相关的复制原点及侧翼序列、立早基因IE180和EP0及潜伏相关转录体转录调节区的甲基化图谱进行测定和比较，确定和验证潜伏感染期病毒DNA复制与转录调节相关区域的甲基化图谱。然后，通过体内去甲基化和体外甲基化对转录活性影响试验，测定病毒复制与转录调节相关区域的甲基化对转录的调节作用。最后，通过凝胶迁移滞留试验确定与甲基化位点相关的调控蛋白，研究DNA甲基化调节病毒基因组复制和转录的机制。本项目为研究PRV潜伏感染机制开辟了新的研究思路和途径，为进一步探讨和揭示潜伏感染建立机制奠定基础，为控制和根除PRV感染提供新思路和理论依据。该机理的阐明对于人类疱疹病毒潜伏感染机理的研究和抗病疗亦有借鉴作用。

中文关键词： 伪狂犬病病毒；潜伏感染；甲基化；microRNA；染色质

英文摘要： The project intends to sequence methylation profiles of Pseudorabies virus during latent infection with MeDIP-chip technology (combination of methylated DNA immunoprecipitation with microarray). DNA methylation profiles located within the Replication origin and flanking sequences and IE180/EP0/latency associated transcript (LAT) Transcriptional regulatory regions of Pseudorabies virus during latent infection will be determined and verified by methylation-Specific PCR (MS-PCR). The function of methylation located within the replication and Transcriptional regulatory regions will be determined by measurement of transcriptional changes of methylated genes after treatment with DNA methyltransferase inhibitor of PRV latent pigs in vivo and the detection the function of regulatory regions after treatment with DNA methylase Sss I by useage of Dual-Luciferase reporter gene system in vitro.Proteins which can bind determined methylated DNA regulatory regions will be isolated by electrophoretic Mobility Shift Assay (EMSA).The project aims at the determination of methylation profiles of Pseudorabies virus during latent infection and its function on the establishment and maintain of latency. It help to elucidate the mechanism of Pseudorabies latency.

英文关键词： Pseudorabies virus；latent infection；methylation；microRNA；chromatin