Toll-like receptors (TLRs)are pattern recognition receptors (PRRs) that are important in invertebrate innate immunity for the recognition and elimination of pathogens. Although they were reported in many fishes, Toll-like receptors subfamily contain a large number of members with different functions that need to research in deep. In the present study, the full-length cDNA of TLR3 from the golden pompano, Trachinotus ovatus, was cloned and characterized. The full length of ToTLR3 cDNA was 3710 bp including an open reading frame of 2760 bp encoding a peptide of 919 amino acids. The derived amino acids sequence comprised of 14 leucine-rich repeats (LRR), capped with LRRCT followed by transmembrane domain and cytoplasmic Toll/IL-1R domain (TIR). Multiple sequence alignment and phylogenetic analysis revealed that ToTLR3 shared the highest similarity to the teleost fish and suggested ToTLR3 is fairly conservative in evolution process. Tissues distribution analysis indicated that ToTLR3 showed a tissue-specific variation with high expression in blood and liver. After the fish were stimulated by poly(I:C), flagellin and LPS, ToTLR3 expression in the liver, intestine, blood, kidney, skin and muscle was significantly upregulated in a time-depended manner, especially in immune related tissues such as liver, blood and kidney. Binding assay revealed the specificity of rToTLR3 for pathogen-associated molecular patterns (PAMPs) and bacteria that included Vibrio harveyi, V. vulnificus, V. anguillarum, Photobacterium damselae, Escherichia coli, Aeromonas hydrophila, Staphylococcus aureus and PolyI:C, LPS, Flagellin, and PGN. In addition, a luciferase reporter assay showed that overexpression ToTLR3 significantly increased NF-κB activity. Collectively, our results suggested that ToTLR3 might play an important role as a pattern recognition receptor (PRR) in the immune response towards pathogen infections, and transmiss the danger signal to downstream signaling pathways.

Toll 样受体 (TLR) 是模式识别受体 (PRR)，在识别和消除病原体的无脊椎动物先天免疫中很重要。虽然在许多鱼类中都有报道，但Toll样受体亚科包含大量具有不同功能的成员，需要深入研究。本研究克隆并鉴定了金鲳鱼TLR3的全长cDNA 。ToTLR3全长cDNA为3710 bp，包括一个2760 bp的开放阅读框，编码919个氨基酸的肽。衍生的氨基酸序列由 14 个富含亮氨酸的重复序列 (LRR) 组成，以 LRRCT 为末端，然后是跨膜结构域和细胞质 Toll/IL-1R 结构域 (TIR)。多序列比对和系统发育分析表明，ToTLR3与硬骨鱼具有最高的相似性，表明ToTLR3在进化过程中相当保守。组织分布分析表明，ToTLR3 呈组织特异性变异，在血液和肝脏中高表达。鱼被poly(I:C)、鞭毛蛋白和LPS刺激后，ToTLR3在肝脏、肠道、血液、肾脏、皮肤和肌肉中的表达呈时间依赖性显着上调，尤其是在肝脏、血液和肾脏等免疫相关组织中。结合试验揭示了 rToTLR3 对病原体相关分子模式 (PAMP) 和细菌的特异性，包括哈维弧菌、创伤弧菌、鳗鱼弧菌、达氏发光杆菌、大肠杆菌、嗜水气单胞菌、金黄色葡萄球菌和 PolyI:C、LPS、鞭毛蛋白, 和 PGN。此外，荧光素酶报告基因检测显示过表达ToTLR3NF-κB 活性显着增加。总的来说，我们的研究结果表明，ToTLR3可能作为模式识别受体 (PRR) 在对病原体感染的免疫反应中发挥重要作用，并将危险信号传递到下游信号通路。