Molecular Clone,Sequence Analysis,and Immune Function Of Duck TLR21 Gene

Toll-like receptors(TLRs)are important pattern recognition receptors(PRRs)that recognize pathogen associated molecular patterns(PAMPs),playing a vital role in early responses to infections.In this study,the cDNA sequence of the complete open reading frame(ORF)of duck TLR21(duTLR21)was cloned and reported for the first time,and related studies were performed.Results as follows:1.Molecular cloning,sequence analysis and tissue expression profiling of duTLR21The duTLR21 was cloned successfully using PCR and RACE techniques based on the design of the TLR21 gene sequence of the bird and the cDNA sequence of 3009 bp in length was submitted to NCBI(GenBank accession number: KY829021).Bioinformatic analysis suggested that duTLR21 gene contains a ORF with a length of 2931 bp,encoding 976 amino acids(aa).The aa sequence comparison showed that duTLR21 had the highest similarity with goose TLR21(92%);followed by chicken TLR21 with a similarity of 76%;the similarity of the duTLR21 and crocodile or newt are both higher than fish TLR21;TLR9 had the lowest similarity with duTLR21 is only 28%.The deduced duTLR21 protein contains 21 LRR motifs,a signal peptide,a TM domain,and an intracellular TIR domain.The predicted crystal structures of duTLR21 protein may have two forms: unliganded and bound to ligand,ligand-bound duTLR21 formed a symmetric TLR21-ligand complex,while unliganded duTLR21 was a monomer.The duTLR21 mRNA transcript levels were detected in 15 tissue samples of ducks by qRT-PCR,and results showed that duTLR21 was expressed in various tissues,especially higher in the immuno-related tissues like peripheral blood,spleen,bursa of Fabricius and cecum.2.Immunological activity of duck TLR21Dual-luciferase reporter assays were used to determine whether TLR21 can recognize CpG-ODN and activate its downstream NF-κB.The results showed that CpG-ODN could indeed activate NF-κB via TLR21.qRT-PCR was used to detect the expression of related genes in DEFs treated with 5 kinds of immune stimulators.It was found that 3 different CpG-ODNs could up-regulate the expression of duTLR21,in which CpG-ODN 2395 andCpG-ODN 2007 were stronger than CpG-ODN HC4040,whereas LPS and Poly(I:C)stimulation did not alter duTLR21 mRNA expression levels.At the same time,all three CpG-ODN up-regulate the expression of downstream IL-1β,IL-6 and IFN-α.TLR21 knockdown significantly decreased the expression of IL-1β,IL-6 and IFN-α stimulated by CpG-ODN.To confirm whether duck TLR21 is involved in the antiviral response,duck embryo fibroblasts(DEFs)and ducklings were infected with duck plague virus(DPV),the expression of duTLR21 in the spleen and bursal of the ducklings and the cells were detected by qRT-PCR,as well as IL-1β,IL-6 and IFN-α.We found that in DEFs,DPV led to a significant up-regulation of duTLR21 and IFN-α mRNA levels both at 24 h and 48 h,but IL-1β,IL-6expression levels had no difference.The expression of duTLR21 and IFN-α in the spleen of ducklings was up-regulated at 2,3,4 d.p.i,and there was no significant change in the expression of IL-1β at any of the three time points,IL-6 expression was up-regulated at 3,4d.p.i.The expression of duTLR21,IL-1β,IL-6 and IFN-α in bursa of Fabricius was upregulated at all 3 time points.In order to investigate the effect of duTLR21 on DPV replication in vitro,the duTLR21 expression plasmid or shRNA plasmid were transfected into the DEFs.By qRT-PCR,the DEFs that were transfected with duTLR21 inhibited the DPV replication and the knockdown of endogenous duTLR21 by shRNA significantly promoted DPV replication.