Toll-like receptor (TLR) family plays an important role in innate immunity for detection of and defense against microbial pathogens. In this study, a novel toll-like receptor (HcTLRn) was characterized from freshwater pearl mussel H. cumingii. The complete sequence of HcTLRn was 3725 bp, and the open reading frame (ORF) encoded 718 amino acid residues. Predicted HcTLRn protein possessed seven atypical leucine-rich repeat (LRR) domains, two typical LRR subfamily domains, a C-terminal domain LRR, a transmembrane domain and an intracellular Toll/interleukin-1 (IL-1) receptor domain. Transcripts of HcTLRn were constitutive expressed in the tissues of healthy mussels and were markedly induced in hepatopancreas and gills after lipopolysaccharide (LPS), peptidoglycan (PGN) and polyinosinic polycytidylic acid (ploy I: C) stimulation. Knockdown of HcTLRn in vivo significantly decreased the mRNA levels of TLR pathway transcription factors p65 and p105 as well as antimicrobial peptides (AMPs) including lysozyme (HcLys), theromacin (HcTher), whey acidic protein (HcWAP), LPS-binding protein/bactericidal permeability increasing protein (HcLBP/BPI) 1 and 2 after mussels challenged by LPS. In situ hybridization results showed that HcTLRn mRNA was significantly increased in hemocytes after LPS, PGN and poly I:C stimulation. HcTLRn protein was mainly expressed in hepatopancreas and gills and was significantly increased after LPS stimulation. Moreover, recombinant extracellular domain of HcTLRn (HcTLRn-ECD) proteins could bind to a variety of bacterial and pathogen-associated molecular patterns such as LPS, PGN, and poly I:C in vitro. Subcellular localization results showed that HcTLRn was mainly distributed near the cell membrane and in cytoplasm. Over-expression of HcTLRn activated the NF-κB luciferase reporter in HEK293T cells. Collectively, these results suggested that HcTLRn was a TLR family member that might play an important role in activation of NF-κB signal pathway in Mollusca.

Toll 样受体 (TLR) 家族在先天免疫中发挥重要作用，用于检测和防御微生物病原体。在这项研究中，从淡水珍珠贻贝H. cumingii 中表征了一种新的 Toll 样受体 (HcTLRn). HcTLRn的完整序列为3725 bp，开放阅读框（ORF）编码718个氨基酸残基。预测的 HcTLRn 蛋白具有七个非典型的富含亮氨酸重复 (LRR) 域、两个典型的 LRR 亚家族域、一个 C 端域 LRR、一个跨膜域和一个细胞内 Toll/白细胞介素-1 (IL-1) 受体域。HcTLRn 的转录本在健康贻贝的组织中组成型表达，并在脂多糖 (LPS)、肽聚糖 (PGN) 和聚肌苷酸（策略 I：C）刺激后在肝胰腺和鳃中显着诱导。体内敲除 HcTLRn 显着降低了 TLR 通路转录因子 p65 和 p105 以及抗菌肽 (AMP) 的 mRNA 水平，包括溶菌酶 (HcLys)、theromacin (HcTher)、乳清酸性蛋白 (HcWAP)、贻贝受到 LPS 攻击后 LPS 结合蛋白/杀菌通透性增加蛋白 (HcLBP/BPI) 1 和 2。原位杂交结果显示，LPS、PGN和poly I:C刺激后血细胞中HcTLRn mRNA显着增加。HcTLRn蛋白主要在肝胰腺和鳃中表达，LPS刺激后显着升高。此外，HcTLRn (HcTLRn-ECD) 蛋白的重组胞外域可以在体外结合多种细菌和病原体相关分子模式，如 LPS、PGN 和 poly I:C。亚细胞定位结果表明HcTLRn主要分布在细胞膜附近和细胞质中。HcTLRn 的过度表达激活了 HEK293T 细胞中的 NF-κB 荧光素酶报告基因。总的来说，