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)

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 stimulation of LPS and poly I:C.In vitro,the AjTBK1 transcripts of Japanese eel liver cells were signi-ficantly enhanced to its peak by the treatment of LPS,poly I:C,PGN,or the 106 CFU/mL A.hydrophila,being up to the 15.3-,5.8-,9.3-and 4.7-fold,respectively.Subcellular localization studies showed that AjTBK1 was evenly distributed in the cytoplasm of HEK293 cells in natural state.AjTBK1 was found to aggregate into spots in the cytoplasm upon the stimulation of LPS or poly I:C in HEK293 cells.Additionally,luciferase assays demonstrated that the AjTBK1 overexpression significantly enhanced the activation of NF-κB,AP1,and IFNβ-responsive pro-moters in HEK293 cells,and robustly up-regulated the activation of NF-κB,AP1,and IFNβ-responsive promoter,which were 1.8-,1.2-,and 1.92-fold induced at 24 h,12 h,and 24 h after cotransfection,respectively.These res-ults collectively suggest that AjTBK1 may function as important positive regulation in innate immunity of host against antibacterial and antiviral infection likely via the activation of NF-κB,AP1,and type I IFN signaling path-ways.