草鱼HMGB2b蛋白对GCRV感染免疫应答的动态互作分子及机理解析

高迁移率族蛋白B（HMGB）家族是一类非组蛋白染色质蛋白，在哺乳动物抗病毒免疫应答中具有必不可少的“前哨”作用，然而具有鱼类特色成员的免疫功能机制还不清楚。申请者发现草鱼HMGB2b抑制GCRV增殖，且特异性地对GCRV表现出强烈的亚细胞迁移。在此基础上，本项目建立草鱼HMGB2b融合标签表达细胞系，GCRV感染，蛋白质免疫共沉淀技术分离互作蛋白，通过LC-MS/MS进行鉴定，双分子荧光互补及反向免疫共沉淀验证。然后，进行模拟分子对接分析。揭示其在细胞核和细胞质的互作分子及机理。本研究不仅将阐明具有鱼类自身特点的新型受体HMGB2b在抗病毒免疫应答中的作用和地位，而且为系统阐释鱼类HMGB家族在免疫调控网络中的功能和机制打开突破口。

High mobility group box (HMGB) gene family are non-histone chromatin proteins, functioning as universal sentinels in antiviral immune responses in mammals. However, little is known about the relevant functions in special members in fish. Applicant finds that that grass carp HMGB2b can inhibit propagation of grass carp reovirus (GCRV) and its subcellular localizations are changed post GCRV infection, specially and remarkably. In the present proposal, CiHMGB2b-tag fusion cell lines are constructed and infected by GCRV. Isolates and identifies interaction proteins by protein co-immunoprecipitation and LC-MS/MS techniques. The potential interaction proteins are verified by bimolecular fluorescence complementation and reverse co-immunoprecipitation techniques. At last, CiHMGB2b and interaction molecules model molecular docking. Clarifying the interaction proteins and nucleic acids of CiHMGB2b and the interaction mechanisms in nucleus and cytoplasm. These researches will not only clarify characteristics and functions of piscine HMGB2b in antiviral immune responses, but also serve the systematic clarification on the functions and mechanisms of piscine HMGBs in immune network.