State Key Laboratory of Developmental Biology of Freshwater Fish, Hunan Normal University, Changsha, 410081, China; College of Life Science, Hunan Normal University, Changsha, 410081, China.
State Key Laboratory of Developmental Biology of Freshwater Fish, Hunan Normal University, Changsha, 410081, China; The Second Xiangya Hospital, Central South University, Changsha, 410011, China.
Fish Shellfish Immunol. 2017 Jul;66:360-371. doi: 10.1016/j.fsi.2017.05.035. Epub 2017 May 16.
Melanoma differentiation-associated gene 5 (MDA5) belongs to RIG-I like receptor (RLR) family, which detects cytosolic viral RNA component in immune response. In this study, MDA5 orthologue of black carp (Mylopharyngodon piceus) has been cloned and characterized. The full-length cDNA of black carp MDA5 (bcMDA5) comprises 3244 nucleotides and the predicted bcMDA5 protein contains 984 amino acids. The constitutive transcription of bcMDA5 was extremely low in all the tested tissues, which included gill, skin, muscle, intestine, kidney, spleen, liver and heart. However, bcMDA5 mRNA level was much enhanced in most selected tissues in response to GCRV or SVCV infection. bcMDA5 migrated around 120 KDa in immunoblot and was identified as a cytosolic protein by immunofluorescent staining in both EPC and HeLa cells. Expressing bcMDA5 in EPC cells resulted in the induction of promoter activity of zebrafish IFN3 or fathead minnow IFN. The EPC cells expressing bcMDA5 obtained improved antiviral ability against both SVCV and GCRV. When EPC cells were co-transfected with plasmids expressing bcMDA5 and bcLGP2, the induced IFN expression by bcMDA5 was obviously enhanced. EPC cells expressing both bcMDA5 and bcLGP2 owned much improved antiviral ability than those cells expressing only bcMDA5 or bcLGP2. In general, our data support the conclusion that bcMDA5 plays an important role in the antiviral innate immune response of black carp and bcLGP2 acts as a positive regulator in bcMDA5 mediated signaling.
黑色素瘤分化相关基因 5（MDA5）属于 RIG-I 样受体（RLR）家族，可在免疫反应中检测细胞溶质中的病毒 RNA 成分。在本研究中，克隆并鉴定了草鱼（Mylopharyngodon piceus）的 MDA5 同源物。草鱼 MDA5（bcMDA5）的全长 cDNA 包含 3244 个核苷酸，预测的 bcMDA5 蛋白包含 984 个氨基酸。在所有测试的组织中，bcMDA5 的组成型转录水平均极低，包括鳃、皮肤、肌肉、肠、肾、脾、肝和心脏。然而，bcMDA5mRNA 水平在大多数选定的组织中因 GCRV 或 SVCV 感染而大大增强。免疫印迹中 bcMDA5 迁移约 120 kDa，通过 EPC 和 HeLa 细胞中的免疫荧光染色鉴定为细胞质蛋白。在 EPC 细胞中表达 bcMDA5 导致斑马鱼 IFN3 或胖头鱼 IFN 的启动子活性诱导。表达 bcMDA5 的 EPC 细胞获得了对 SVCV 和 GCRV 的抗病毒能力提高。当 EPC 细胞共转染表达 bcMDA5 和 bcLGP2 的质粒时，bcMDA5 诱导的 IFN 表达明显增强。表达 bcMDA5 和 bcLGP2 的 EPC 细胞比仅表达 bcMDA5 或 bcLGP2 的细胞具有更好的抗病毒能力。总体而言，我们的数据支持以下结论：bcMDA5 在草鱼抗病毒先天免疫反应中发挥重要作用，bcLGP2 在 bcMDA5 介导的信号转导中起正调控作用。
