猪源转录因子AP-2δ通过增强rep基因启动子活性促进猪圆环病毒2型的复制  

Porcine transcription factor AP-2δ promotes porcine circovirus type 2 replication through enhancing the activity of the rep gene promoter

作　　者：王越[1] 宋东峰 林翠 李佳容 王胜男 顾金燕[1] 周继勇[2] Yue Wang;Dongfeng Song;Cui Lin;Jiarong Li;Shengnan Wang;Jinyan Gu;Jiyong Zhou(Institute of Immunology, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China;Key Laboratory of Animal Virology of Ministry of Agriculture, Zhejiang University, Hangzhou 310058, Zhejiang, China)

机构地区：[1]南京农业大学免疫研究所,江苏南京210095 [2]浙江大学农业部动物病毒学重点实验室,浙江杭州310058

出　　处：《生物工程学报》2018年第12期1985-1995,共11页Chinese Journal of Biotechnology

基　　金：国家科技支撑计划(No.2015BAD12B01);国家自然科学基金(No.31402198);中央高校基本科研业务费(Nos.KJQN201524;KYZ201730);江苏高校优势学科建设资助~~

摘　　要：本研究旨在探究猪圆环病毒2型(Porcine circovirus type 2, PCV2)能否借助rep基因启动子区的转录因子结合位点调控自身基因转录,并寻找参与这一调控过程的宿主因子。凝胶迁移实验证实rep基因启动子具有核蛋白结合活性。DNA-pull down联合液相-串联质谱分析鉴定到了可与rep基因启动子结合的猪源转录因子AP-2δ(Porcine transcription factor AP-2δ, poTFAP2δ)。双荧光素酶报告基因试验、实时荧光定量PCR、免疫印迹及间接免疫荧光等试验证明poTFAP2δ不仅可以特异性地增强rep基因启动子活性,而且可以在PCV2感染过程中促进rep和cap基因的转录、翻译及PCV2病毒滴度。文中揭示了PCV2利用宿主蛋白促进自身增殖的分子机制,为进一步从病毒与宿主互作角度阐明PCV2的致病机制提供新的研究思路,亦为PCV2高效疫苗的研制提供理论基础。Several putative transcription factor binding sites (TFBSs) exist in the PCV2 rep gene promoter. To explore if porcine circovirus type 2 (PCV2) could regulate the viral replication by using these TFBSs, we conducted electrophoretic mobility shift assay (EMSA), DNA-pull down and liquid chromatography-tandem mass spectrometric (LC-MS/MS) assays. EMSA confirmed the binding activity of the rep gene promoter with nuclear proteins of host cells. DNA-pull down and LC-MS/MS identified the porcine transcription factor AP-2δ (poTFAP2δ) could bind the PCV2 rep gene promoter. Dual-luciferase reporter assay, quantitative real-time PCR, Western blotting and indirect immunofluorescent assay demonstrated that poTFAP2δ could not only promote the activity of the rep gene promoter, but also enhance the transcription/translation activity of the rep/cap gene and the virus titer of PCV2 during the entire life cycle of PCV2 infection. This study revealed the molecular mechanism of PCV2 using host proteins to enhance the viral replication, provided a new perspective for studying the pathogenic mechanism of PCV2 from virus and host interactions, and provided a theoretical basis for developing highly effective PCV2 vaccines.

关 键 词：猪圆环病毒2型 rep基因启动子 转录因子结合位点 猪源转录因子AP-2δ 

分 类 号：S852.651[农业科学—基础兽医学]