Molecular cloning and immune function analysis of MyD88 gene in Anguill anguilla

Myeloid differentiation factor 88(MyD88)is a universal and crucial adaptor protein of signal transduction activity elicited by TLR(toll-like receptor)superfamily and plays an essential role in the innate immunity response.In this study, a MyD88 gene, AaMyD88, was cloned for the first time from Anguilla anguilla. Its full-length cDNA sequence is 1 539 bp, with an 846 bp open reading frame encoding a protein of 282 aa.The predicted three-dimensional structure of AaMyD88 can be merged well with the crystal structure of the human MyD88 and has the typical death domain and TIR(toll-like/IL-1 receptor)domain of the MyD88 family.In the TIR domain, three conserved boxes, named box 1, box 2 and box 3, are found.According to the comparison with known MyD88 amino acid sequences, the putative protein of AaMyD88 showed the highest identities with Ictalurus punctatus(76.3%), and other bony fishes(67.5%-73.2%), but low identities(61.6%-62.6%)with mammalian.All the sequences were clustered into three main branches in which all MyD88 of fish clustered together, while the MyD88 of mammalians and amphibians formed two separate clusters.qRT-PCR revealed the expression of AaMyD88 could be detected in all adult tissues examined.The highest expression level was observed in the liver, followed by the heart, intestine, and spleen, and the lowest expression level was found in muscle and gills.After injection with goat IgG, the AaMyD88 expression level was obviously up-regulated in the kidney at 7 d and returned to the normal level after 14 d.The obvious up-regulation of the AaMyD88 expression level was observed in the spleen from 7 d to 21 d, peaking at 7 d with a value 1.7 times higher than that of the kidney.After stimulation with poly I:C, AaMyD88 transcripts in fin cell line were down-regulated significantly at 3 h, increased strongly from 6 h to 48 h, and peaked at 12 h.After treatment with LPS, the expression level of AaMyD88 in fin cell line was sharply down-regulated at 3 h and began to significantly increase at 6 h and peaked at 12 h.As the time progressed past this time point, the expression level rapidly decreased to the control levels at 24 h post-treatment.Compared with the group of LPS treatment, the expression level of AaMyD88 in the poly I:C group was observed continuously higher from 12 h to 48 h.Our results suggested that AaMyD88 might play important roles during the immune response of Anguilla anguilla against the different pathogen microbes.