日本鳗鲡TBK1基因的克隆与免疫功能分析  

Molecular cloning and immune functional analysis of TBK1 gene in Japanese eel(Anguilla japonica)

作　　者：徐元凯 彭欣慰 林鹏[1,3,4] 王艺磊 冯建军[1,3,4] XU Yuankai;PENG Xinwei;LIN Peng;WANG Yilei;FENG Jianjun(Fisheries College,Jimei University,Xiamen 361021,China;Ningbo Institute of Oceanography,Ningbo 315832,China;Engineering Research Centre of Eel Modern Technical Industry,Ministry of Education,Xiamen 361021,China;Key Laboratory of Healthy Mariculture for the East China Sea,Ministry of Agriculture and Rural Affairs,Xiamen 361021,China)

机构地区：[1]集美大学水产学院,福建厦门361021 [2]宁波海洋研究院,浙江宁波315832 [3]鳗鲡现代产业技术教育部工程研究中心,福建厦门361021 [4]农业农村部东海海水健康养殖重点实验室,福建厦门361021

出　　处：《水产学报》2023年第12期157-173,共17页Journal of Fisheries of China

基　　金：福建省自然科学基金(2020J01671);鳗鲡现代产业技术教育部工程研究中心开放基金(RE202110);农业农村部东海海水健康养殖重点实验室开放基金(2020ESHML02)。

摘　　要：为了阐明鱼类TANK结合激酶1(TBK1)在免疫应答密切相关的NF-κB、I型IFN及MAPK信号通路中的调控作用,本实验通过cDNA末端快速扩增技术(SMART RACE)从日本鳗鲡中克隆了TBK1基因cDNA全长序列,命名为AjTBK1,利用实时荧光定量PCR(qRT-PCR)检测了在体和离体状态下不同病原体相关分子模式(PAMPs)及嗜水气单胞菌对日本鳗鲡AjTBK1基因表达水平变化的影响,通过构建绿色荧光蛋白pEGFP-TBK1和pCMV-TBK1真核表达质粒对AjTBK1亚细胞定位以及AjTBK1过表达对NF-κB、AP-1、IFN-β启动子荧光素酶活性的激活作用进行研究。蛋白质序列分析显示,日本鳗鲡AjTBK1编码731个氨基酸,其三维丝带空间结构与人类TBK1相似,具有保守的激酶结构域(KD)、泛素样结构域(ULD)、二聚化支架结构域(SDD)以及C端结构域(CTD),在系统发育树中与其他鱼类TBK1家族聚为一支。qRT-PCR检测发现AjTBK1在多种组织中广泛表达,且在肝脏和肠中高表达。经LPS、poly I:C、嗜水气单胞菌免疫注射后,AjTBK1基因表达水平在日本鳗鲡肝脏中显著提高,而肾脏中的表达量则在LPS和poly I:C刺激后显著降低。离体实验中,经LPS、poly I:C、PGN以及不同浓度嗜水气单胞菌刺激后的日本鳗鲡肝脏细胞AjTBK1基因表达水平均有显著升高。亚细胞定位结果显示,天然状态下的AjTBK1在HEK293细胞质中分布,经LPS和poly I:C刺激后呈聚集点状分布。此外,双荧光素酶活性检测发现过表达的AjTBK1可显著增强NF-κB、AP-1和IFN-β启动子荧光素酶活性。以上研究表明,AjTBK1可以通过激活NF-κB、AP-1和I型IFN信号通路,在机体抗细菌和抗病毒先天免疫应答中发挥重要的调控作用。As an important serine/threonine kinase in the IKK family,TANK Binding Kinase 1(TBK1)plays a critical role in innate immune response by activating NF-κB and type I IFN signaling pathways in mammals.Although several studies have reported that fish TBK1 was involved in the regulation of type I IFN production,information on TBK1's close association with antimicrobial immune responses in the regulation of NF-κB and MAPK signaling pathways is still limited in teleost fish.In order to elucidate the regulation of fish TBK1 in NF-κB,I IFN,and MAPK immune response signaling pathways,the full-length cDNA of a TBK1 homologue,AjTBK1,was cloned by SMART RACE from Japanese eel,and its characteristics of expression in response to vari-ous PAMPs and Aeromonas hydrophila infection were investigated both in vivo and in vitro by quantitative real-time polymerase chain reaction(qRT-PCR).In addition,the subcellular localization of AjTBK1 GFP fusion pro-tein and the induction of AjTBK1 overexpression in the activation of NF-κB,AP1 and type I IFN performed by Dual-Glo luciferase assay system were also detected.Amino acid sequence analysis indicated that AjTBK1 encodes a polypeptide of 731 amino acids,which has the conserved N-terminal kinase domain(KD),a ubiquitin-like domain(ULD),a scaffold dimerization domain(SDD),and a C-terminal domain(CTD).The predicted three-dimensional structure of AjTBK1 is similar to that of human TBK1,and AjTBK1 is clustered with other fish famil-ies in the phylogenetic tree.Quantitative real-time PCR(qRT-PCR)analysis revealed that AjTBK1 is broadly expressed in a wide range of tissues,with high expression in liver and intestine.In vivo,the expression of AjTBK1 in the liver of Japanese eel was significantly increased by 1.8-fold at 6h,1.8-fold at 6 h,and 1.9-fold at 48 h after injection with LPS,viral mimic poly I:C and A.hydrophila infection,respectively.The AjTBK1 expression in kid-ney was found to increase at 12 h and 24 h with 1.9-and 1.6-fold after A.hydrophila infection,but decreased fol-lowing the

关 键 词：日本鳗鲡 TANK结合激酶1(TBK1) 信号通路 亚细胞定位 双荧光素酶活性 

分 类 号：Q785[生物学—分子生物学] S942.5[农业科学—水产养殖]