Molecular Cloning And Fuction Studies On Goose TLR3and5Gene

Frequent outbreak of infectious diseases causes huge economic losses in gooseindustry, due to little is known about the anti-infection mechanism of immune system andthe absence of a better prevention techniques in this species. To elucidate the role of Tolllike receptor (TLRs) in the process of anti-infective immunity of goose may not onlyimprove the preventative or treatmental methods to special disease, but collected angenetic material for molecular anti-disease breeding. In present study, the reversetranscription polymerization chain reaction (RT-PCR) and PCR amplification of cDNAends (Smart RACE) methods were used to clone the full-length cDNA of goose TLR3andTLR5, and predicted the molecular function according putative protein. We aslo designedthe quantitative PCR primers to detect the expression of TLRs mRNA in various tissuesand constructed an eukaryotic expression vector of TLRs and transfected to HEK293Tcells, the activity of nuclear factor-kappa B is detected by double fluorescent reportergene system after TLRs transfected and special ligand excited. Meanwhile, fluorescencequantitative PCR method were used to detect the TLRs and correlated pro-cytokines inPBMC that cultured in vitro and excited by different pathogens. The results showed that:（1）The TLR3full length cDNA of goose exhibited2874bp in length, including a5′-terminal (UTR) of149bp, a3′-terminal (UTR) of34bp and an open reading frame(ORF) of2691bp, encoding an protein of896amino acides, in which calculated molecularmass is102.13kDa, a theoretical pI is8.53, and have a putative signal secretion peptidelocated in27aa of its N-terminal region. TLR3consist of a Toll/interleukin-receptor (TIR)domain, a transmembrane domain and14leucine-rich repeats (LRRs) domain. Theleucine-rich domain (LRR) and interleukin-1receptor domain (TIR) were conserved ingoose TLR3compared to other species. Homology alignment of TLR3and its TIRdomains revealed that shared high percentage of identities with chicken (84.6%). Byquantitative RT-PCR, we founded that the TLR3gene of goose was broadly expressed inall tested tissues, and highestly expressed in Pancreatic gland, Spleen and Duodenum. Theincrease of NF-κB promoter activity by Poly i:c was dependent the TLR3expression. Theexpression of TLR3, IL-6and IFN-γ mRNA, but not IL-1β, was upregulated significantlyafter Poly i:c and avian flu virus (H5N1) stimulated in PBMC of goose that cultured invitro. (2) The full length cDNA of goose TLR5exhibited2967bp in length, including a5′-terminal (UTR) of215bp, a3′-terminal (UTR) of384bp and an open reading frame(ORF) of2583bp, that encode a protein of860amino acides and has a calculatedmolecular mass of98.88kDa, a theoretical pI of6.34and a putative signal secretionpeptide located in22aa of its N-terminal region. Structurally, TLR5has aToll/interleukin-receptor (TIR) domain, a transmembrane domain and7leucine-richrepeats (LRRs) domain. Homology alignment of TLR5and its TIR domains revealed thatgoose TLR5shared highest percentage of identities with Gallus Gallus TLR15(81.3%).By quantitative RT-PCR, we founded that the TLR5gene of goose was broadly expressedin all tested tissues, but highestly expressed in kidney and spleen. The increase of NF-κBpromoter activity by Flagellin was dependent the TLR5expression in293T cell.Salmonella and Flagellin stimulate upregulated the expression of TLR3, IL-8and IL-1βmRNA significantly in PBMC of goose that cultured in vitro.We concluded that we cloned the full length cDNA of TLR3and TLR5in goosesuccessfully. The combining with the ligands of TLR3/5indued the upregulation ofpro-cytokines expression in in vitro cultured PBMC throuth NF-kappaB pathway. TLR3and TLR5play an important role in anti-infection of avian flu virus and Salmonella ingoose.