Molecular Cloning, Expression Of Chicken TBK1 Gene And Its Effect On IRF3 Signaling Pathway

TRAF family member-associated NF-κB activator (TANK)-binding kinasel (TBK1), also known as NFκB activating kinase (NAK) or TRAF2-associated kinase (T2K), was identified as important adaptor molecules linking upstream receptor signals to downstream gene activation in many pathways such as inflammatory, apoptotic and immune responses. Recently TBK1 was proposed to be a hub protein which is implicated in various pathogenic pattern recognition receptors (PRRs) in innate immune. The importance of TBK1 in antiviral immunity is well established in mammal model, but in chicken, the molecular characterization and potential function of TBKl is still unclear. Thus, we choose chicken TBK1 as the target gene. In present study, we cloned and characterized the full-length coding sequences of chicken TBK1 and detected its tissue expression profile in chicken by qRT-PCR. We also detected the differential expression of chicken TBK1 with or without ALV-J or virus analog challenges, both in vitro and in vivo. To verify whether the chicken TBK1 are positive regulatory gene of IRF3, IFNβ and IFNa gene, we determined the mRNA expression of TBK1, IRF3, IFNβ and IFNa by qRT-PCR, after stimulating cells by ALV-J infection or poly I:C transfection in the presence of siRNA. The results are as follows:(1) We obtain the full-length coding sequence of chicken TBK1. Sequencing results revealed that the ORF of chicken TBKl consists of 2190 base pair (bp) fragment which encoding a deduced protein of 729 amino acid residues. Multiple sequence alignment analysis exhibited that chicken TBKl sequence similarity to other birds and mammals, and own at least 85% homology to other mammals, which indicate that it is conserved during evolution. Conserved domain analysis of the chicken TBKl protein was found in N-terminus, including the protein kinase domain and the ubiquitin-like domain; besides, ATP-binding site and S172 are located in the kinase domain.(2) Quantitative real-time PCR (qRT-PCR) results showed chTBKl was ubiquitously expressed in chicken tissues and especially highly expressed in immune tissues such as in spleen, thymus and bursa of Fabricius (P<0.01).(3) The expression of chTBKl was significantly up-regulated by infected with avian leukosis virus subgroup J (ALV-J) in chicken spleen, thymus and bursa of Fabricius (P< 0.05 or P<0.01). The expression of chTBKl was significantly up-regulated by infected with avian leukosis virus subgroup J (ALV-J) or polyinosinic-polycytidylic acid (polyI:C) in chicken embryo fibroblasts (CEFs) (P<0.01). The results indicate that TBK1 is involved in anti-virus immune in chicken and chicken cell.(4) Consistent with the activation of chTBK1, the interferon regulatory factor 3 (IRF3) and IFNβ gene in CEFs were also up-regulated after challenged with ALV-J or polyI:C(P<0.05 or P< 0.01). In contrast, the expression of IRF3 and IFNβ in CEFs were significantly reduced by siRNA targeting chTBK1 in comparison with negative control (NC) during ALV-J infection or polyl:C transfection (P<0.05 or P<0.01). This indicated that TBK1 plays positive role in the expression of IRF3, IFNP and IFNa.