Molecular characterization and expression analysis of Toll-like receptor 21 cDNA from Paralichthys olivaceus

Toll-like receptor (TLR) is believed to play crucial role in host defense of pathogenic microbes in innate immune system. In the present study, the full-length cDNA of Paralichthys olivaceus Toll-like receptor 21 (Po-TLR21) was cloned by homology cloning and rapid amplification of cDNA ends (RACE) technique. The Po-TLR21 cDNA sequence was 3687 bp, containing an open reading frame of 2922 bp encoding 973 amino acids. TMHMM and SMART program analysis indicated that protein contained one transmembrane domain, eighteen leucine-rich repeats (LRRs), and one Toll/IL-1 receptor homology domain (TIR). Multiple alignment analysis of the Po-TLR21 protein-coding sequence with other known TLR21 from grouper, pufferfish, zebrafish, cod, catfish, carp and chicken showed the homology of 67%, 63%, 54%, 52%, 51%, 49%, and 39%, respectively. The Po-TLR21 mRNA expression patterns were measured by real-time PCR. The results revealed that TLR21 is widely expressed in various tested healthy tissues, and highly expressed in spleen and gill. In vivo immunostimulation experiments revealed that expression of TLR21 is modulated by Vibrio anguillarum (V. anguillarum), CpG oligodeoxynucleotides (CpG ODN) and poly I:C. Moreover, the inhibitor of homodimerization of myeloid differentiation factor 88 (MyD88) could significantly reduce the up-regulation of TLR21, MyD88, and tumor necrosis factor (TNF) expression in CpG ODN or poly I:C-treated head kidney cells in vitro. These results indicate that TLR21 may be involved in the pathogen recognition in the early innate immune.