Signaling Functions And Anti-infection Effects Of Chicken TLR5 And Adaptor Protein MyD88

Toll-like receptors(TLRs)are the earliest discovered family of mammalian innate immune pattern recognition receptors(PRRs)and the most widely studied pattern recognition receptors.Their research has greatly enriched the knowledge of immune biology.TLRs and other PRRs recognize pathogen-associated molecular patterns(PAMPs)and can activate cellular signaling pathways to produce a variety of inflammatory cytokines,chemokines and type I interferons(IFNs),which can quickly launch a series of antimicrobial immune responses.This study mainly investigates the signaling functions and anti-infection effects of chicken TLR5(chTLR5)and it's signaling adaptor protein chMyD88,in an attempt to provide new scientific clues for further research on TLR5 in poultry.This research mainly includes the following aspects:1 chTLR5 and chMyD88 gene cloning,expression and signal function identificationThe chTLR5 and chMyD88 protein-encoding gene sequences were amplified by RT-PCR from chicken HD11 and DF1 cells,respectively.chTLR5 was 2605bp and chMyD88 was 919bp.The PCR products were ligated to the intermediate vector pENTR4-MCS-3FLAG,respectively,and recombinant pENTR4 vectors were subjected to the LR site-specific recombination with the target vector pDEST47 to successfully construct eukaryotic expression plasmids pcDNA-chTLR5-3FLAG and pcDNA-chMyD88-3FLAG.Western blotting showed that chTLR5 and chMyD88 exhibited high protein expression in transfected 293T cells,with sizes of approximately 120kD and 35kD,respectively.NF-?B promoter dual luciferase reporter gene and qRT-PCR tests showed that chTLR5 expression has weak constitutive NF-?B activity and can activate low-level gene expression of inflammatory cytokines IL-1? and IL-8;stimulation with bacterial flagellin can further enhance NF-?B activation and inflammatory gene expression downstream of chTLR5.chMyD88 expression has a strong constitutive activity,which trigger downstream NF-?B activation and induces significant gene expression of the inflammatory cytokines TNF-? and IL-8.The above results demonstrate that both chTLR5 and chMyD88 possess signal activity.2 Detection of chTLR5 and chMyD88 mRNA tissue distribution by quantitative RT-PCRBased on the chicken TLR5 and MyD88 sequences obtained,quantitative PCR primers were designed respectively to establish chTLR5 and chMyD88 fluorescent quantitative RT-PCR methods,and to analyze the TLR5 and MyD88 mRNA expression levels in different chicken tissues.The results showed that real-time quantitative RT-PCR had good sensitivity,and specificity.chTLR5 mRNA is widely expressed in various tissues,with the highest expression in the pancreas and cecum,whereas chMyD88 is also highly expressed in multiple tissues,with the highest in the pancreas and liver.Both mRNAs of chTLR5 and chMyD88 are highly expressed in immune organ spleen,indicating the critical role of spleen.3 Anti-infection effects of exogenous chTLR5 and chMyD88Intermediate recombinant vectors pENTR4-chTLR5-3FLAG and pENTR4-chMyD88-3FLAG were subjected to LR site-specific recombination with the vector plenti-CMV DEST puro to obtain the recombinant viral expression vector.The validated expression vectors plenti-CMV-TLR5-3FLAG,plenti-CMV-MyD88-3FLAG,and empty vector plenti-CMV were transfected into chicken macrophage HD11 cells,respectively,followed by infections with Salmonella typhimurium(S.T),Vesicular stomatitis virus(VSV-GFP)or Newcastle disease virus(NDV-RFP).Quantitative RT-PCR results showed that compared with the control HD11 cells,the proliferation levels of S.T in chTLR5 KO and chMyD88 KO HD11 cells were reduced significantly,while the expression levels of related cytokine genes increased.The VSV and NDV replication levels in chTLR5 and chMyD88 transfected HD11 cells were both reduced.The fluorescence intensity of the viruses observed by the fluorescence microscope was consistent with the above results from qRT-PCR.In addition,chTLR5 and chMyD88 transfected cells had increased expression levels of downstream antiviral and inflammatory cytokine genes after viral infections.4 Anti-infection effects of endogenous chTLR5 and chMyD88Using CRISPR-Cas9 technology,gRNAs corresponding to chTLR5 and chMyD88 were designed,and gRNAs were ligated to lentiviral vector pLenti-CRISPRV2 to obtain recombinant gRNA viral vectors.The packaged lentiviruses were used to infect chicken HD11 cells to obtain the knockout stable HD11 cell lines after puromycin selection.The constructed chTLR5 KO,chMyD88 KO and vector control HD11 stable cells were used for infection analysis with S.T,VSV-GFP or NDV-RFP.The results showed that compared with the control cells,the proliferation levels of S.T in chTLR5 KO and chMyD88 KO HD11 cells were increased significantly,while the expression levels of related cytokine genes decreased.The VSV and NDV replications increased in chTLR5 KO and chMyD88 KO HD cells,and the expressions of antiviral and inflammatory cytokine genes were accordingly decreased to varying degrees.The above results suggest that endogenous chTLR5 and chMyD88 play a certain defense role when host cells are attacked by pathogens including bacteria and viruses.5 chMyD88 prokaryotic expression and preparation of monoclonal antibodyThe intermediate vector pENTR4-chMyD88-3FLAG was subjected to LR site-specific recombination with the vector pDEST527 to obtain a recombinant prokaryotic expression vector pDEST527-chMyD88-3FLAG.The expression plasmid was transformated into E.coli DE3/-BL21 to induce protein expression,and the correct protein expression was confirmed by Western blotting.The optimal conditions were selected to purify chMyD88 soluble protein from bacterial inclusion bodies.The chMyD88 purified protein was injected into 4?5 week old BALB/c mice for immunization,and the mice spleen cells were fused with myeloma cells.The hybridoma cell supernatant was detected for specific antibodies by ELISA to obtain positive clones.In Western blotting analysis,the monoclonal antibody form the positive clone was able to detect both exogenous and endogenous chMyD88 proteins.The chMyD88 monoclonal and polyclonal antibodies acquired in this study provide useful experimental materials and tools for chMyD88 protein-related functional studies.