Objective  Gene sequence and polyclonal antibodies of myeloid differentiation factor 88 (MyD88) in Larus ridibundus were studied to reveal the protein structure and functions for further research on the innate immune signaling pathway.

Method Total RNA was extracted from the peripheral blood lymphocytes of L. ridibundus using the trizol method. MyD88 was amplified by RT-PCR. The similarity, genetic relationship, physiochemical properties, secondary and tertiary structures of the gene were analyzed using bioinformatics software. Prokaryotic expression strain of MyD88 recombinant protein was obtained by transformation with the induction conditions optimized and purified by Ni-column, ultrafiltration and gradient dialysis followed by emulsification with Freund's adjuvant to immunize New Zealand white rabbits in preparing the polyclonal antibody serum. Titer of the obtained polyclonal antibody was determined by two-way agar diffusion assay, and specificity against MyD88 recombinant protein identified by SDS-PAGE and western blotting.

Result  The CDS region of MyD88 was 921 bp encoding 306 amino acids. The similarity of the gene with that of Rissa tridactyla was the highest at 98.8%, while the lowest at 84.6% with that of black-headed gull and mallard. It had a molecular size of approximately 34.5 kDa that contained one N-glycosylation site and 24 phosphorylation sites. There was no signal peptide and transmembrane structure. The secondary structure of the MyD88 protein was mainly random coil, while the tertiary structure, consistent with the secondary structure. The hydrophilic protein consisted of a complete death domain and TIR domain. The expression of the 54 kDa prokaryotic protein peaked in 4 h at 37 ℃ in 1.0 mmol·L⁻¹ IPTG. The prepared polyclonal antibody serum had a titer of 1∶16 that could effectively recognize the recombinant MyD88 protein of L. ridibundus indicating high in specificity.

Conclusion A hydrophilic protein, MyD88 encoded rich in phosphorylation sites with a complete death domain and TIR domain. The constructed recombinant protein could be expressed in large quantities in vitro exerting strong immunogenicity, and the prepared polyclonal antibodies were highly specific.