Immunobiological Function Of Porcine TLR5 And Its Anti-Infection Application

The innate immune system is the first line of defense against pathogens by continuously monitoring pathogen-associated molecular patterns(PAMPs)to activate a range of defense mechanisms to eliminate infection.Innate immune cells express various pattern recognition receptors(PRRs)that are responsible for recognizing PAMPs of microorganisms.Toll-like receptors(TLRs)are an important class of PRRs that play an important role in the innate immune system.As a member of the TLR family,TLR5 is able to recognize bacterial flagellin.Flagellin binds to TLR5 and relies on the adaptor protein MyD88 for signal transduction,which can activate the NF-?B and MAPK signaling pathways,leading to the production of downstream inflammatory cytokines and chemokines,and plays an important role in the body's resistance to bacterial infection.Some studies have also shown that flagellin may also inhibit virus replication after activating TLR5.In this study,porcine TLR5(pTLR5)was cloned from pig-derived cells,and a eukaryotic expression plasmid was constructed to identify its protein expression and signaling function.In addition,pTLR5 and pMyD88 gene knockout cell lines and pTLR5 over-expressing cell lines were constructed,together with transfected cells,all these cells were used to study pTLR5 and its signaling adaptor pMyD88 for their cell signaling functions and anti-microbial effects.The main research contents are as follows:1 Gene cloning,protein expression and signal function of pTLR5RT-PCR was used to amplify pTLR5 gene from total RNA of PAM cells,and a recombinant pENTR4-pTLR5-3FLAG intermediate shuttle plasmid was constructed.The confirmed pENTR4-pTLR5-3FLAG intermediate plasmid were subjected to site-specific recombination(LR)reaction with destination vecotr pDEST47 to obtain the recombinant pcDNA-pTLR5-3FLAG eukaryotic expression plasmid.The pTLR5 protein expression in transfected 293T cells was measured by Western Blotting,and the size of pTLR5 was observed about 100KDa.As shown by quantitative RT-PCR,pTLR5 can mediate bacterial flagellin stimulation in transfected cells,causing the expressions of downstream genes IFN-?and IL-8.2 The roles of pTLR5 and pMyD88 against swine E.coliThe CRISPR-Cas9 technology was used to design the gRNAs of pTLR5 and pMyD88.The gRNAs were ligated with pCRISPRv2 lentiviral vectors to obtain recombinant gRNA viral plasmids.PK15 cells were infected with packaged gRNA-expressing lentivirus,and a stable knockout(KO)cell line was obtained after selection with 2?g/mL puromycin.At the same time,the pENTR4-pTLR5-3 FLAG plasmid were performed to LR reaction with pLentiCMV DEST puro to obtain the lentiviral expression plasmid pLentiCMV-pTLR5-3FLAG.PK15 cells were infected by the packaged pTLR5 expressing lentivirus,and pTLR5 overexpression(OE)PK15 stable cell line was obtained after puromycin selection.In addition,the constructed expression plasmid pEGFP-pMyD88 was transfected alone or co-transfected with pcDNA-pTLR5-3FLAG to PK15 cells.The above cells were stimulated with flagellin and porcine Escherichia coli O20,respectively.Quantitative RT-PCR was used to detect the downstream gene expression of the cells and the bacterial colony counting was used to analyze the bacterial growth.In TLR5 KO and MyD88 KO PK15 cells,the levels of flagellin and E.coli induced downstream genes including inflammatory factors and interferon-stimulated genes(ISG)were decreased,while the bacterial growth increased significantly.In TLR5 and MyD88 OE cells,flagellin and E.coli activated downstream genes including inflammatory factors and interferon-stimulated gene expression levels were increased,but the bacterial growth was significantly reduced.3 The roles of pTLR5 and pMyD88 against Salmonella typhimuriumCRISPR gRNA expressing lentiviruses were used to infect PAM cells and the infected PAMs were selected with 1?g/mL puromycin to obtain pTLR5 KO PAM and MyD88 KO PAM stable cell lines.At the same time,pTLR5-3FLAG lentivirus was used to infect PAM cells,and pTLR5 OE PAM stable cell line was obtained after puromycin selection.In addition,the pEGFP-pMyD88 was transfected alone or co-transfected with pcDNA-pTLR5-3FLAG into PAM cells.The above cells were stimulated with flagellin and Salmonella typhimurium(S.T),respectively.Quantitative RT-PCR was used to detect the downstream gene expression of the cells and bacterial colony counting was used to analyze the bacterial growth.In TLR5 KO and MyD88 KO PAM cells,flagellin and ST induced downstream genes including inflammatory factors and ISG expression levels were reduced,while the bacterial growth increased significantly.In TLR5 and MyD88 OE PAM cells,flagellin and S.T activated downstream genes including inflammatory factors and ISG expression levels were increased,but the bacterial growth was significantly reduced.4 The roles of pTLR5 and pMyD88 in anti-swine influenza virusThe above pTLR5 KO,pMyD88 KO stable PAM cells,pTLR5 OE stable PAM cells,pMyD88 transfected PAMs,and pTLR5 and pMyD88 co-transfected PAMs were used for stimulation by infection with swine influenza virus H9N2-C1,respectively.Quantitative RT-PCR was used to detect downstream gene expression and H9N2 viral gene replication.In both pTLR5 KO and pMyD88 KO stable PAM cells,the expression levels of downstream genes were decreased,and the expression of viral HA and M genes increased.In pTLR5 OE stable PAM cells and pMyD88-transfected PAM cells,the expression levels of downstream genes induced by the virus were increased,and the replication level of the virus H9N2 decreased.In the PAM cells co-transfected with pTLR5 and pMyD88,the virus replication level was further reduced compared with single transfected cells.These results suggest that pig TLR5-MyD8 8 not only has antibacterial effect,but also exerts antiviral function.