College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009 Jiangsu, PR China.
Dev Comp Immunol. 2013 Dec;41(4):766-71. doi: 10.1016/j.dci.2013.07.018. Epub 2013 Aug 2.
Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) have recently been identified as cytoplasmic sensors for RNA virus. Recent research has shown that RIG-I, a member of this family, play an important role in innate immunity. In this study, we cloned the RIG-I gene from Jinding duck by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). We determined that the cDNA of duRIG-I contains a 14-bp 5' UTR, a 2802-bp open reading frame, and alternative 3' UTRs (295-bp and 927-bp) and encodes a polypeptide of 933 amino acids. Based on this sequence, the duRIG-I protein is predicted to have conserved domains typical of RLRs. In addition, duRIG-I was found to be distributed throughout DF1 cells by indirect immunofluorescence, as predicted. duRIG-I mRNA was scarcely detected in healthy tissues by semi-quantitative RT-PCR (sqRT-PCR). To study the role of RIG-I in innate immunity, we used synthetic double-stranded RNA to mimic viral infection in vivo and detected duRIG-I transcripts in spleen and liver by quantitative real-time PCR (qRT-PCR). The expression of duRIG-I mRNA was significantly elevated at 8h post-injection (P < 0.05) and was indistinguishable from control levels at other time points (P > 0.05). These results suggest that duRIG-I plays an important role in innate immune responses to double-stranded RNA viruses and warrant further studies to reveal the possible mechanism.
维甲酸诱导基因 I(RIG-I)样受体(RLRs)最近被鉴定为细胞质中 RNA 病毒的传感器。最近的研究表明,该家族成员 RIG-I 在先天免疫中发挥重要作用。在这项研究中,我们通过反转录聚合酶链反应(RT-PCR)和快速扩增 cDNA 末端(RACE)从金定鸭中克隆了 RIG-I 基因。我们确定 duRIG-I 的 cDNA 含有一个 14 个碱基对的 5'UTR、一个 2802 个碱基对的开放阅读框和替代的 3'UTR(295 个碱基对和 927 个碱基对),并编码一个 933 个氨基酸的多肽。基于该序列,duRIG-I 蛋白被预测具有 RLRs 的典型保守结构域。此外,间接免疫荧光法预测,duRIG-I 分布在 DF1 细胞中。半定量 RT-PCR(sqRT-PCR)检测发现 duRIG-I mRNA 在健康组织中几乎检测不到。为了研究 RIG-I 在先天免疫中的作用,我们使用合成的双链 RNA 模拟体内病毒感染,并通过定量实时 PCR(qRT-PCR)检测脾和肝中的 duRIG-I 转录物。注射后 8 小时(P < 0.05)duRIG-I mRNA 的表达显著升高,其他时间点与对照水平无差异(P > 0.05)。这些结果表明,duRIG-I 在对双链 RNA 病毒的先天免疫反应中发挥重要作用,值得进一步研究以揭示可能的机制。