Immunobiological Function Of Pig NOD1 And NOD2 And The Application

During the growth and development,mammals are constantly threatened by pathogenic microorganisms in the external environment,so they have evolved a established immune system to resist the infection of external pathogens.The immune system can be divided into the innate immune system and the acquired immune system The innate immune system is the first line of defense against the invasion of foreign pathogenic factors,mainly through the recognition of highly conserved pathogen-associated molecular patterns(PAMPs)by its pattern recognition receptors(PRRs),which then activates a immune response through a series of signal cascades in the cell.Recent studies have shown that NODI and NOD2,the representatives of the pattern recognition receptors NOD-like receptors(NLRs)protein family,can initiate the innate immune response via recognizing their respective specific ligands iE-DAP and MDP,and play a role in the body’s immune defense against bacterial infection by activating downstream signaling pathways such as NF-κB and MAPKs;Recently reports also showed that they can recognize and resist viral infections,although the specific mechanisms are still unclear.With the development of animal husbandry nowaday,pigs are both important economic animals and promising model animals.The health of pigs is closely linked to the needs of human life.Therefore,in this study,NODI and NOD2 receptor-encoding genes were cloned from porcine-derived cells and eukaryotic expression plasmids were constructed to identify two receptor protein expression and signaling functions.In addition.the NOD1 and NOD2 knockout cell lines were constructed to explore their important role in the fight against pathogenic microorganisms;Finally,monoclonal antibody of porcine NODI was prepared by immunizing mice with the purified protein from bacteria,which wil be a useful tool for further research.The specific research contents are divided into the following three parts:1 Cloning,expression and signal function of porcine NOD1(pNOD1)and NOD2(pNOD2)genesThe full-length cDNA sequences of the two receptor-encoding genes pNOD1 and pNOD2 were amplified by RT-PCR from pig PAM and PK15 cells,with pNOD1 2867 bp and pNOD23039 bp.The eukaryotic expression plasmids pcDNA-pNOD1 and pcDNA-pNOD2 carrying the 2×HA or 3×FLAG tags at the C-terminus were successfully constructed using Gateway LR cloning technology.The protein expression of pNOD1 and pNOD2 were examined by Western blotting.The sizes of proteins were about 125kDa;the protein expression level of pNOD1 was high,but the expression level of pNOD2 protein was low.The pNOD1 and pNOD2 signaling functions were examined as following:the eukaryotic expression plasmid was co-transfected into HEK293T cells with ELAM(NF-κB)promoter firefly luciferase reporter gene and actin promoter Renilla fluorescein reporter gene.The transfected cells were stimulated with ligands iE-DAP and MDP,respectively.The dual luciferase reporter system assay showed that pNOD1 has strong constitutive signaling activity,and both pNOD1 and pNOD2 can respond to the corresponding specific ligand and mediate downstream NF-κB signal activation.At the same time,quantitative RT-PCR was used to detect the downstream gene expression of pNOD1 and pNOD2 transfected and stimulated HEK293T cells.The results showed that the transcription level of downstream cytokine IL-8 was significantly increased,which was consistent with the activation of downstream NF-κB signal.Analysis of the signaling relationship between pNOD1 and pNOD2 using the NF-κB promoter luciferase reporter gene assay showed that no significant mutual inhibition or promotion was found.In addition,pNOD2 was transfected into NF-κB dual luciferase HEK293 reporter cells,and the pNOD2-NF-KB dual luciferase stable reporter cells were obtained by G418 selection.The successfully constructed pNOD2-NF-κB dual luciferase reporter cells serve as an effective system for future screening efforts.2 The roles of anti-pathogenic microorganisms by pNODl and pNOD2The guide RNAs(gRNAs)corresponding to the pNOD1 and pNOD2 coding genes were designed by CRISPR-cas9 technology,and the gRNAs were ligated with the lentiviral vector pLentiCRISPRv2 with the recombinant gRNA vectors confirmed by sequeccing.The pNOD1 and pNOD2 expression plasmids and the corresponding gRNA vectors were co-transfected into HEK293T cells,and the validity and specificity of gRNAs were ensured by Western blotting.The selected gRNA plasmids were subjected to lentiviral packaging,the viral supernatants were used to infect PAM,PK15 and IPEC-J2 cells,respectively,and the gene knockout(KO)stable cell lines were obtained after screen with puromycin.The constructed pNOD1,pNOD2 KO and control stable cell lines were used for infection with Salmonella typhimurium(PAM),pig Escherichia coli(PK15),swine and human influenza viruses(PAM),and porcine epidemic diarrhea virus(IPEC-J2).Compared with the control cells,bacterial counting and qRT-PCR both showed that the bacterial growth and viral replication levels were significantly increased in the pNOD1 KO and pNOD2 KO cell lines.In particular,with the infection of Salmonella typhimurium and influenza viruses,the transcription levels of the downstream antiviral protein genes were also significantly decreased.These demonstrate the importance of pNOD1 and pNOD2 in host defense when it is infected by the pathogens.3 Expression and purificaiton of pNOD1 from bacteria and preparation of pNOD1 monoclonal antibodyThe prokaryotic expression plasmid pDest527-pNOD1 was constructed by Gateway LR cloning technology and transformed into Escherichia coli DE3/BL21.The correct expression of the target protein was detected by Western-blotting,and the expression of recombinant pNOD1 protein was mainly in the bacterial precpitation as a inclusion body.The large amount of recombinant bacteria were cultured and induced by IPTG for pNOD1 expression using the optimized conditons.The inclusion bodies were purified from the bacterial pellet,and denatured by 8M urea,and soluble protein solution was renatured sequentially with the 6M,4M.2M and OM urea containing buffer to obtain soluble pNOD1 protein.BALB/c mice were immunized with purified pNOD1 protein,and the serum antibody titer of immunized mice was 1:10000-1:50000 by indirect ELISA.The spleen cells of the immunized mice and the myeloma SP2/0 cells were fused by PEG1500.The hybridoma cells were subjected to multiple ELISA tests,and ELISA positive clones subjected for three rounds of subcloning by limited dilution method.Finally a positive hybridoma cell strain stably secreting antibody was obtained.The monoclonal antibody secreted by this cell line can detect the expression of both exogenous and endogenous pNOD1 protein by Western blotting,thus a useful tool for future functional study.