State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Science, Hunan Normal University, Changsha 410081, China.
State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Science, Hunan Normal University, Changsha 410081, China.
Fish Shellfish Immunol. 2017 Oct;69:108-118. doi: 10.1016/j.fsi.2017.08.016. Epub 2017 Aug 15.
Tank-binding kinase 1 (TBK1) plays a pivotal role in the induction of type I IFNs in higher vertebrates. To explore the function of TBK1 in teleost, TBK1 of black carp (Mylopharyngodon Piceus) was cloned and characterized in this paper. The full-length cDNA of black carp TBK1 (bcTBK1) consists of 2857 nucleotides and the predicted bcTBK1 protein contains 727 amino acids, which includes an N-terminal kinase domain (KD), an ubiquitin-like domain (ULD) and two C-terminal coiled-coils. The transcription of bcTBK1 was constitutively detected in all the selected tissues and bcTBK1 mRNA level was increased in all selected tissues in response to SVCV or GCRV infection except that in muscle post GCRV invasion. The transcription of bcTBK1 in Mylopharyngodon Piceus fin (MPF) cells was up-regulated by the stimulation of SVCV, GCRV or poly (I:C) but not by LPS treatment. bcTBK1 migrated around 80 kDa in immunoblot assay and was identified as a cytosolic protein by immunofluorescence staining. bcTBK1 showed strong IFN-inducing ability in reporter assay and presented strong antiviral activity against both GCRV and SVCV in EPC cells. The reporter assay demonstrated that TRAF6 of black carp (bcTRAF6) up-regulated bcTBK1-induced IFN expression and the subcellular distribution of bcTBK1 overlapped with that of bcTRAF6 when these two proteins were co-expressed in EPC cells. Taken together, our study support the conclusion that bcTBK1 plays an important role in the antiviral innate immune response of black carp against SVCV and GCRV, in which its activity was positively regulated by bcTRAF6.
Tank-binding kinase 1 (TBK1) 在高等脊椎动物中诱导 I 型干扰素的产生中发挥着关键作用。为了探索 TBK1 在硬骨鱼类中的功能，本文克隆并鉴定了草鱼（Mylopharyngodon piceus）的 TBK1。草鱼 TBK1（bcTBK1）的全长 cDNA 由 2857 个核苷酸组成，预测的 bcTBK1 蛋白包含 727 个氨基酸，其中包括一个 N 端激酶结构域（KD）、一个泛素样结构域（ULD）和两个 C 端卷曲螺旋。bcTBK1 的转录在所有选定的组织中均持续存在，并且在 SVCV 或 GCRV 感染后，除肌肉外，所有选定的组织中的 bcTBK1 mRNA 水平均增加。SVCV、GCRV 或 poly（I:C）刺激可上调草鱼 Mylopharyngodon piceus 鳍（MPF）细胞中 bcTBK1 的转录，但 LPS 处理不能上调其转录。免疫印迹分析显示，bcTBK1 迁移约 80 kDa，免疫荧光染色鉴定其为细胞质蛋白。bcTBK1 在报告基因检测中具有很强的 IFN 诱导能力，并在 EPC 细胞中对 GCRV 和 SVCV 表现出很强的抗病毒活性。报告基因检测表明，草鱼 TRAF6（bcTRAF6）上调 bcTBK1 诱导的 IFN 表达，并且当这两种蛋白在 EPC 细胞中共表达时，bcTBK1 的亚细胞分布与 bcTRAF6 的分布重叠。综上所述，我们的研究支持以下结论：bcTBK1 在草鱼抗病毒先天免疫反应中发挥重要作用，抵抗 SVCV 和 GCRV，其活性受到 bcTRAF6 的正向调节。
