Cloning and expression analysis of a Toll-like receptor 21 (TLR21) gene from turbot, Scophthalmus maximus

Abstract

Toll-like receptor 21 (TLR21) is a non-mammalian TLR recognizing unmethylated CpG DNA and considered as a functional homolog of mammalian TLR9. In the present study, a TLR21 gene was cloned from turbot, Scophthalmus maximus, its immune responsive expression was subsequently studied in vivo. The turbot (Sm)TLR21 gene is an intronless gene with a length of 3527 bp and encodes a peptide of 984 amino acids. The deduced protein possesses a signal peptide sequence, a leucine-rich repeat (LRR) domain composed of 16 LRR motifs, a transmembrane (TM) region and a Toll/interleukin-1 receptor (TIR) domain. Phylogenetic analysis grouped it with other teleost TLR21s. Quantitative real-time PCR (qPCR) analysis demonstrated the constitutive expression of SmTLR21 mRNA in all twelve examined tissues with higher levels in the lymphomyeloid-rich tissues like spleen and head kidney. Further, upon stimulation with polyinosinic: polycytidylic acid [poly(I:C)], turbot reddish body iridovirus (TRBIV) and CpG oligodeoxynucleotides (CpG-ODN) 2395, the SmTLR21 mRNA expression was up-regulated in the gills, head kidney, spleen and muscle. The maximum increases of SmTLR21 transcript levels ranged from 1.3 to 8.1-fold and appeared at 3 h to 5 day post-injection depending on different organs and stimuli. These findings suggest that SmTLR21 may play an important role in the immune responses to the infections of a broad range of pathogens that include RNA and DNA viruses and bacteria.