Characterization, expression and function analysis of the TLR3 gene in golden pompano (Trachinotus ovatus).

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.

Molecular characterization of coding sequences and analysis of Toll-like receptor 3 mRNA expression in water buffalo (Bubalus bubalis) and nilgai (Boselaphus tragocamelus).

Toll-like receptor 3 (TLR3), an antiviral innate immunity receptor recognizes double-stranded RNA, preferably of viral origin and induces type I interferon production, which causes maturation of phagocytes and subsequent release of chemical mediators from phagocytes against some viral infections. The present study has characterized TLR3 complementary DNA (cDNA) in buffalo (Bubalus bubalis) and nilgai (Boselaphus tragocamelus). TLR3 coding sequences of both buffalo and nilgai were amplified from cultured dendritic cell cDNA and cloned in pGEMT-easy vector for characterization by restriction endonucleases and nucleotide sequencing. Sequence analysis reveals that 2,715-bp-long TLR3 open reading frame encoding 904 amino acids in buffalo as well as nilgai is similar to that of cattle. Buffalo TLR3 has 98.6 and 97.9% identity at nucleotide level with nilgai and cattle, respectively. Likewise, buffalo TLR3 amino acids share 96.7% identity with cattle and 97.8% with nilgai. Non-synonymous substitutions exceeding synonymous substitutions indicate evolution of this receptor through positive selection among these three ruminant species. Buffalo and nilgai appear to have diverged from a common ancestor in phylogenetic analysis. Predicted protein structures of buffalo and nilgai TLR3 from deduced amino acid sequences indicate that the buffalo and nilgai TLR3 ectodomain may be more efficient in ligand binding than that of cattle. Furthermore, TLR3 messenger RNA expression in tissues as quantified by real-time PCR was found higher in nilgai than buffalo.