Functional Study Of Pattern Recognition Receptor NLRC In Innate Immune Signaling Pathway Of Common Carp(Cyprinus Carpio)

NLRs(Nucleotide-binding oligomerization domain,NOD-like receptors)are an important class of pattern recognition receptors(PRRs),which exist in the cytoplasm and can recognize a variety of intracellular pathogen-associated molecular patterns(PAMPs),activate the corresponding signaling pathways,and finally induce the production of cytokines,thereby exerting the function of resisting pathogens.Three NLR subfamilies,NLR-A,NLR-B,and NLR-C,were identified in teleosts by analysis of non-mammalian vertebrate databases.Among them,NLR-C is a kind of NLRs unique to teleosts.Its Nterminus is FISNA,RING or PYRIN domain with different structural characteristics,and its C-terminus is rich in Leucine-rich repeat(LRR).Contains an additional B30.2(PRYSPRY)domain.Studies have found that the B30.2 domain may play an important role in the recognition of pathogens.The current research on fish NLR-C is mainly limited to the distribution of tissue expression and the expression changes in tissues after pathogenic stimulation,while the research on the immune mechanism is relatively less.In this study,carp was used as the research object,and the immune mechanism of carp NLRC in the NLR-C family was systematically studied.The main research contents and results of this paper are as follows:1.An ORF fragment of an NLR-C subfamily gene was identified in carp,and the gene was named carp NLRC.Sequence analysis showed that the ORF region of carp NLRC gene was 3075 bp,encoding a total of 1024 amino acids.Through domain prediction,it was found that the N-terminal had a special FISNA domain,and the Cterminal LRR domain contained an additional B30.2 domain.It can be seen from the phylogenetic tree that carp NLRC is in a different branch from other animal NLRs,and belongs to the same branch as other teleost NLR-C family genes.Through protein sequence similarity comparison,it was found that it had the highest similarity with the NLRC3-like protein of the golden-haired barb.In this study,we identified ORF fulllength sequences of ASC,RIP2 and MAVS genes,respectively named as CcASC,CcRIP2 and CcMAVS.Sequence analysis showed that the ORF region of CcASC was 600 bp,the ORF region of CcRIP2 was 1752 bp,and the ORF region of CcMAVS was 1749 bp.Through protein sequence similarity comparison,it was found that both CcASC and CcRIP2 had the highest similarity with goldfish,while CcMAVS had the highest similarity with golden-haired catfish.The results of real-time fluorescent quantitative PCR showed that carp NLRC,CcASC,CcRIP2 and CcMAVS mRNA expression levels in carp healthy tissue head kidney,spleen,skin,gills,foregut,midgut,hindgut,liver and muscle There were differential expressions in all tissues,among which the relative expression levels of carp NLRC and CcMAVS were the highest in the head kidney;the relative expression level of CcASC was the highest in the gills;the relative expression level of CcRIP2 was the highest in the spleen.2.To study the function of carp NLRC to activate downstream signaling pathways.Firstly,construct eukaryotic expression vectors of carp NLRC and downstream signaling molecules,conduct laser confocal experiments to confirm that carp NLRC is stably expressed in the cytoplasm,and conduct Western blot to confirm the expression of CcASC and CcRIP2 and CcMAVS in HEK293T cells.Through the dual luciferase reporter gene experiment(Firefly&Renilla-Glo Luciferase),the results showed that the overexpression of carp NLRC in HEK293T cells can enhance the activity of the luciferase reporter gene of nuclear transcription factor NF-κB(Nuclear factor kappa-B).In order to further explore the downstream signaling molecules of carp NLRC,we simultaneously co-transfected carp NLRC and downstream signaling molecules CcASC,CcRIP2 and CcMAVS into HEK293T cells,and detected the effect on the activation of transcription factor NF-κB by Luciferase assay.The results showed that carp Co-transfection of NLRC with CcRIP2 and CcMAVS could significantly enhance the activation of NF-κB,while co-transfection with CcASC decreased the activity of NF-κB.3.Explore the ligands recognized by carp NLRC and the regulation of cytokines in carp EPC cells.After the carp NLRC eukaryotic expression vector was transferred into EPC cells,the expression levels of downstream cytokines were detected by fluorescent quantitative PCR.The results showed that in normal EPC cell culture state,the expression levels of IFN-1,Viperin,ISG15 and PKR genes were significantly up-regulated compared with those transfected with empty plasmid.In addition,after stimulating the EPC cells overexpressing carp NLRC with pathogen-associated molecular patterns,including LPS,Poly I:C,PGN,LTA,i E-DAP and MDP,the results of qRT-PCR showed that although INF-1,Viperin,ISG15,PKR Compared with the cells transfected with empty plasmid,the EPC cells transfected with carp NLRC had a tendency to up-regulate INF-1,ISG15,and PKR mRNA before and after Poly I:C stimulation compared with the EPC cells transfected with empty plasmid The upregulation trend of these genes was much enhanced before and after Poly I:C stimulation.In addition,the up-regulated folds of INF-1 and Viperin gene expression levels in EPC cells transfected with carp NLRC before and after LPS stimulation were much larger than those in EPC cells transfected with empty plasmid before and after LPS stimulation.These results suggest that carp NLRC plays a key role in both antiviral and antibacterial innate immunity,it may recognize Poly I:C and LPS,and regulate antiviral-related cytokines INF-1,Viperin,ISG15 and PKR effect.