College of Animal Science and Technology, Northwest A&F University, Shaanxi Key Laboratory of Molecular Biology for Agriculture, Yangling 712100, China.
Fish Shellfish Immunol. 2010 Apr;28(4):712-8. doi: 10.1016/j.fsi.2010.01.009. Epub 2010 Jan 28.
The cytoplasmic helicase protein MDA5 (melanoma-differentiation-associated gene-5) recognizes long molecules of viral double-stranded RNA (dsRNA) and single-stranded RNA with 5' triphosphate and mediates type I interferon secretion. In the present study, the first MDA5 gene in fish was cloned and characterized from grass carp Ctenopharyngodon idella. The full length of the C. idella MDA5 (CiMDA5) cDNA is 3233 nucleotides in length and encodes a polypeptide of 961 amino acids. The deduced amino acid sequence contained six main structural domains: a CARD (caspase activation and recruitment domain), a DEXDc (DEAD/DEAH box helicase domain), a ResIII (conserved restriction domain of bacterial type III restriction enzyme), two HELICc (helicase superfamily c-terminal domain) and an RD (regulatory domain). The CiMDA5 mRNA was widespread expression in the tested tissues, was high level in spleen, skin and gill tissues, and was up-regulated by GCRV injection by semi-quantitative RT-PCR assay. The CiMDA5 transcripts in spleen were significantly up-regulated at 12 h (1.80 folds, P < 0.05), reached the crest at 24 h (7.48 folds, P < 0.05), and then recovered to normal level at 48 h post-injection (P > 0.05) of grass carp reovirus (GCRV). In liver, the temporal expression of CiMDA5 mRNA was significantly increased at 48 h (5.00 folds, P < 0.05) and returned to control level at 72 h (P > 0.05) after GCRV challenge. These data implied that CiMDA5 involved in the early stage of antiviral innate immune defense to GCRV in grass carp, and provided new insight into the evolution research of RLR (RIG-I like receptor) gene family.
细胞质解旋酶蛋白 MDA5（黑色素瘤分化相关基因-5）识别病毒双链 RNA（dsRNA）和 5'三磷酸单链 RNA，并介导 I 型干扰素的分泌。本研究首次从草鱼 Ctenopharyngodon idella 克隆并鉴定了鱼类的 MDA5 基因。C. idella MDA5（CiMDA5）cDNA 的全长为 3233 个核苷酸，编码 961 个氨基酸的多肽。推导的氨基酸序列包含六个主要结构域：CARD（半胱天冬酶激活和募集结构域）、DEXDc（DEAD/DEAH 盒解旋酶结构域）、ResIII（细菌 III 型限制酶的保守限制结构域）、两个 HELICc（解旋酶超家族 C 端结构域）和一个 RD（调节结构域）。CiMDA5 mRNA 在检测的组织中广泛表达，在脾脏、皮肤和鳃组织中表达水平较高，并用半定量 RT-PCR 检测到 GCRV 注射后上调。草鱼呼肠孤病毒（GCRV）感染后，脾脏中 CiMDA5 转录物在 12 h 时显著上调（1.80 倍，P < 0.05），24 h 时达到峰值（7.48 倍，P < 0.05），48 h 时恢复正常水平（P > 0.05）。在肝脏中，CiMDA5 mRNA 的时间表达在 GCRV 攻击后 48 h 时显著增加（5.00 倍，P < 0.05），并在 72 h 时恢复到对照水平（P > 0.05）。这些数据表明，CiMDA5 参与了草鱼对 GCRV 的抗病毒先天免疫防御的早期阶段，并为 RLR（RIG-I 样受体）基因家族的进化研究提供了新的见解。
