Abstract：
Toll-like receptors (TLRs) are pattern recognition receptors that trigger host immune responses against various pathogens by detecting evolutionarily conserved pathogen-associated molecular patterns (PAMPs). TLR21 is a member of the Toll-like receptor family, and emerging data suggest that it recognises unmethylated CpG DNA and is considered a functional homologue of mammalian TLR9. However, little is known regarding the role of TLR21 in the fish immune response. In the present study, we isolated the cDNA sequence of TLR21 from the largemouth bass (Micropterus salmoides) and termed it MsTLR21. The MsTLR21 gene contained an open reading frame (ORF) of 2931 bp and encodes a polypeptide of 976 amino acids. The predicted MsTLR21 protein has two conserved domains, a conserved leucine-rich repeats (LRR) domain and a C-terminal Toll-interleukin (IL) receptor (TIR) domain, similar to those of other fish and mammals. In healthy largemouth bass, the TLR21 transcript was broadly expressed in all the examined tissues, with the highest expression levels in the gills. After challenge with Nocardia seriolae and polyinosinic polycytidylic acid (Poly[I:C]), the expression of TLR21 mRNA was upregulated or downregulated in all tissues tested. Overexpression of TLR21 in 293T cells showed that it has a positive regulatory effect on nuclear factor-kappaB (NF-κB) and interferons-β (IFN-β) activity. Subcellular localisation analysis showed that TLR21 was expressed in the cytoplasm. We performed pull-down assays and determined that TLR21 did not interact with myeloid differentiation primary response gene 88 (Myd88); however, it interacted with TIR domain-containing adaptor inducing interferon-β (TRIF). Taken together, these findings suggest that MsTLR21 plays important roles in TLR/IL-1R signalling pathways and the immune response to pathogen invasion.
摘要：
Toll样受体(TLR)是模式识别受体,其通过检测进化上保守的病原体相关分子模式(PAMP)来触发针对各种病原体的宿主免疫应答。TLR21是Toll样受体家族的成员,新出现的数据表明，它识别未甲基化的CpGDNA，被认为是哺乳动物TLR9的功能同源物。然而,关于TLR21在鱼类免疫反应中的作用知之甚少。在本研究中,我们从大嘴鲈鱼（Micropterussalmoides）中分离出TLR21的cDNA序列，并将其命名为MsTLR21。MsTLR21基因包含2931bp的开放阅读框（ORF），并编码976个氨基酸的多肽。预测的MsTLR21蛋白具有两个保守结构域，一个保守的富含亮氨酸重复序列（LRR）域和一个C端Toll-白介素（IL）受体（TIR）域，与其他鱼类和哺乳动物相似。在健康的大嘴鲈鱼中，TLR21转录本在所有检查的组织中广泛表达，在ill中表达水平最高。用诺卡氏菌和聚肌苷酸聚胞苷酸（聚[I：C]）挑战后，在所有受试组织中,TLR21mRNA的表达上调或下调.TLR21在293T细胞中的过表达表明，它对核因子-κB（NF-κB）和干扰素-β（IFN-β）活性具有正调节作用。亚细胞定位分析表明TLR21在细胞质中表达。我们进行了下拉测定，并确定TLR21不与骨髓分化原代反应基因88（Myd88）相互作用；然而，它与含有TIR结构域的衔接子相互作用，诱导干扰素-β（TRIF）。一起来看,这些发现表明MsTLR21在TLR/IL-1R信号通路和病原体入侵的免疫应答中起重要作用。
