Inhibitor of nuclear factor kappa-B kinase subunit alpha (IKKα) plays a pivotal role in the activation of nuclear factor kappa-B (NF-κB) pathway in response to pathogens infections in mammals, but the information about IKKα in the regulation of immune responses is still limited in teleost fishes. In the present study, the full-length cDNA of an IKKα homologue, AjIKKα, was cloned by 5′ and 3′ SMART RACE from Japanese eel, and its characteristics of expression in response to various PAMPs and A. hydrophila infection were investigated both in vivo and in vitro using quantitative real-time polymerase chain reaction (qRT-PCR). In addition, the subcellular localization of AjIKKα GFP fusion protein and the induction of AjIKKα in the activation of NF-κB, type I IFN and AP1 performed using Dual-Glo luciferase assay system were also detected. Sequence comparison analysis revealed that AjIKKα has typical conserved domains, including an N-terminal kinase domain, an ubiquitin-like domain, a scaffold dimerization domain, and a C-terminal NEMO-binding domain. The predicted three-dimensional structure of AjIKKα is similar to that of human IKKα. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed a broad expression for AjIKKα in a wide range of tissues, with the highest expression in the liver, followed by the intestine, gills, and spleen, and with a lower expression in the muscle and heart. The AjIKKα expressions in the liver and kidney were significantly induced following injection with the viral mimic poly I:C and Aeromonas hydrophila infection, whereas the bacterial mimic LPS down-regulated the expression of AjIKKα in the spleen. In vitro, the AjIKKα transcripts of Japanese eel liver cells were significantly enhanced by the treatment of LPS, poly I:C, CpG-DNA, and PGN or the stimulation of different concentration of Aeromonas hydrophila (1 × 10⁶ cfu/mL, 1 × 10⁷ cfu/mL, and 1 × 10⁸ cfu/mL). Luciferase assays demonstrated that AjIKKα expression could significantly induce NF-κB, AP-1 and type I IFN promoter activation in a dose-dependent manner. Additionally, subcellular localization studies showed that AjIKKα was evenly distributed in the cytoplasm in the natural state, but AjIKKα was found to aggregate into spots in the cytoplasm after the stimulation of LPS and poly I:C. These results collectively indicated that AjIKKα plays an important role in innate immunity of host against antibacterial and antiviral infection likely via the activation of NF-κB, AP1and type I IFN signaling pathway.

核因子κB激酶亚基的抑制剂（IKKα）在响应哺乳动物病原体感染时在激活核因子κB（NF-κB）途径中起关键作用，但有关IKKα在免疫调节中的信息硬骨鱼类的反应仍然有限。在本研究中，IKKα同源物的全长cDNA，AJ IKKα，用5'和3'从日本鳗鲡SMART RACE克隆，其响应于各种PAMP和表达的特征嗜水感染都进行了研究使用定量实时聚合酶链反应（qRT-PCR）在体内和体外进行。另外，Aj的亚细胞定位IKKαGFP融合蛋白和诱导AJ IKKα在NF-κB的活化，I型IFN和AP1使用双Glo萤光素酶测定系统中也检测到执行的。序列比较分析表明，AJ IKKα具有典型保守结构域，包括N末端激酶结构域，泛素样结构域，二聚化的支架结构域和C-末端NEMO结合域。的预测的三维结构AJ IKKα是类似于人的IKKα。实时定量聚合酶链反应（qRT-PCR）分析显示Aj的广泛表达IKKα在广泛的组织中，在肝脏中表达最高，其次是肠，腮和脾脏，在肌肉和心脏中表达较低。该AJ在肝脏和肾脏IKKα表达均显著诱导注射后与病毒模拟物聚I：C和嗜水气单侵染，而未细菌LPS模拟下调的表达AJ在脾IKKα。在体外，该AJ日本鳗鲡肝细胞IKKα转录由LPS的治疗进行显著增强，聚I：C，CpG的DNA，和PGN或不同浓度的刺激嗜水（1×10 ⁶  CFU /毫升， 1×10⁷  cfu / mL和1×10 ⁸  cfu / mL）。萤光素酶测定显示，AJ IKKα表达可诱导显著NF-κB，AP-1和I型IFN的启动子激活以剂量依赖的方式。此外，亚细胞定位研究表明，AJ IKKα均匀分布在自然状态下的细胞质中，但AJ IKKα被发现聚集成在LPS刺激后胞浆点和聚I：C。这些结果共同表明，AJ IKKα在针对抗菌和抗病毒感染宿主的先天免疫中起重要作用通过NF-κB的活化可能，AP1and I型IFN信号传导途径。