Molecular Cloning, Bioinformatics Analysis And Immune Function Of Goose TLR7 And TLR21

Toll like receptors (TLRs), which are the so-called pattern recognition receptors, mediate the host innate immune response and further adaptive immune after binding the invading pathogens. In this study, we successfully cloned the full-length of Sichuan white goose TLR7 and TLR21 cDNA. Bioinformatics analysis suggested that goose TLR7 has a complete open-reading-frame (ORF) with 1045 aa, thus signal peptide sited in position 1 to 27, transmembrane domain sited in position 836 to 860, and the homology Toll/IL-1 Receptor (TIR) domain sited in position 884 to 1034. Goose TLR21 also has a complete ORF contained 975 aa. Moreover, the signal peptide is sited in position 1 to 35, transmembrane domain is sited in position 757 to 779 and the conserved TIR domain is sited in position 809 to 951. When compared the amino acid alignments of TLR7 and TLR21 from different animals, we found that the more affinity between two species showed the more identity in the sequences.To figure out the tissue distribution of TLR7 and TLR21 in goose, semi-quantitative RT-PCR and quantitative RT-PCR were performed.β-actin and GAPDH served as reference genes to quantitate and recorrect the result. We found that the mRNA expression level of goose TLR7 was highest in bursa of Fabricius, and immune-associated tissues like cecum, Hadrian gland also showed an up-regulation. Interestingly, the abundantly up-regulated TLR7 in lung may lead to anti-respiratory disease. When compared the TLR7 mRNA expression in immune-associated tissues between adult goose and gosling, we found that bursa of Fabricius and spleen both have a high level expression. The immune response is based on bursa of Fabricius and thymus in gosling, while adult goose is dependent on period blood, bursa of Fabricius and spleen. Maybe, the evolutions of goose dose change the immune function in tissues. TLR21 also shows an immune-associated tissue dependent result, such as Hadrian gland and thymus. When compared goose TLR21 mRNA expression in whole tissues between adult goose and gosling, we found a similar result that goose TLR21 expression level got down-regulated in bursa of Fabricius. In general, TLR7 and TLR21 were highly expression in immune-associated tissues which was important for the function. The different results in tissue between adult goose and gosling may suggest that the immune system of goose would get mature along with body developemnt.To figure out the immune function of goose TLR7 and TLR21, some agonists were chosen in this study. R848, Imiquimod, Poly(LC) and ODN2006 are well-known TLRs ligands, and lead to down-stream immune response. R848 is both TLR7 and TLR8 ligand, Imiquimod is only TLR7 ligand, Poly(I:C) is TLR3 ligand and ODN2006 is TLR9 and TLR21 ligand. Agonists treated goose spleen mononuclear cells (MNCs) and goose peripheral blood mononuclear cells (PBMCs) were detected according to quantitative RT-PCR, with PBS group as a negative control. TLR7 and TLR21 were detected in the research by qRT-PCR, as well as the down-stream pro-inflammatory cytokines IL-6, IL-1β. type I interferon IFN-a and type II interferon IFN-y. β-actin and GAPDH served as reference genes to quantitate and recorrect the result. We found that R848 and Imiquimod both can enhance goose TLR7 and the down-steam cytokines IL-6, IL-1 β, and IFN-a as expected. ODN2006 could up-regulate the mRNA expression of goose TLR21, also led to an up-regulation in IL-6, IL-1β, IFN-α and IFN-y. As a positive control, neither goose TLR7 nor goose TLR21 could be induced by Poly(I:C). But the down-stream cytokines IL-6 and IFN-a got enhanced which may give an insight into another receptor TLRS (not been reported). In general, these agonists all can bind TLRs and lead to innate immune response in goose MNCs and goose PBMCs. The results provided an new sight in choosing immunologic adjuvant preventing goose from virus infection.To figure out the anti-virus roles of goose TLR7 and TLR21, we chose new type gosling viral enteritis virus (NGVEV) to challenge goose MNCs and goose PBMCs, with PBS group as a negative control. The goose TLR7 and TLR21 were detected by quantitative RT-PCR to ensure the correct induced innate immune signaling pathway. Also IL-6, IL-1β, IFN-a and IFN-y were studied to reflect the down-steam immune response. β-actin and GAPDH served as reference genes to quantitate and recorrect the result. We found that upon NGVEV challenge, goose TLR7 showed a normal expression level on Oh, 24h,48h,72h,96h. But the up-regulation of down-stream cytokines on 24h suggested the correct immune response exist which led us to another receptors. Actually, we found goose TLR21 get up-regulated on transcriptional level upon NGVEV challenge 24h then. Whether goose TLR21 can be induced by NGVEV? We detected the TLR21-related down-stream cytokines IL-6, IL-1β, IFN-a and IFN-y with the same sample. Finally, we noticed that all the related cytokines get significant up-regulated. The result suggested that goose TLR21 can be induced by NGVEV and ODN2006. Namely, goose TLR21 is essential to anti-NGVEV research and ODN2006 is hopeful becoming an immunologic adjuvant preventing goose from NGVEV infection.In conclusion, we lay a foundation of goose anti-virus innate immune receptor research, and provide a feasible process to prevent and control goose disease in this study.-...