A Study On The Immunorecognition Of IE-DAP From Gram-Gegative Bacteria By Miichthys Miiuy NOD1

In the process of growth and development,vertebrates are continue to be infected by different pathogenic microorganisms.To resist the invasion of pathogens,vertebrates have evolved a series of immune system through the long evolutionary progress.This immune system was an important way for the organism to combat disease,and can divide into the Innate immune system and the Adaptive immunity system.The innate immune system is the first line to defense against pathogen invasion.It can be activated after recognize the pattern of recognition-associated molec?lar patterns(PAMPs)by various pattern-recognition receptors(PRRs).Then through signal cascade to induce the immune response.NLRs(Nucleotide-binding domain and leucine-rich repeat containing)are an important intracell?lar pattern recognition receptors,they can recognize a variety of PAMPs and activate inflammatory responses.Therefore,NLRs attract more attention in recent years.NOD1(Nucleotide-binding oligomerization domain-containing protein 1)is one of important members of NLR family.In mammals,NOD1 can activate the immune response by recognize specific PAMPs to protect the organism.In lower vertebrates,Adaptive immunity system is not yet complete,so the innate immune system will plays an essential role in preventing the occurrence of diseases.In this paper,a simple study was conducted to explore the pathogens that can be recognized by NOD1,and the signal pathways that activated by NOD1.In addition,the domains and the functions of NOD1 were also be explored.The res?lts of this study are as follows:(1)qRT-PCR was carried out to detect NOD1 expression in the tissues and cells of miiuy croaker(Miichthys miiuy).We found that the expression of NOD1 was significantly increased in Liver tissue that was infected by V.anguillarum and in macrophage that were terated by PGN and iE-DAP,so we guess that NOD1 may play an important role in resisting the invasion of Gram-negative bacteria.(2)Through dual-luciferase reporter gene analysis,we found that NOD1 can promote the expression of NF-?B reporter plasmid,but does not have a effect on theexpression of other reporter genes plasmids.This suggests that miiuy croaker NOD1 may induce immune response by activating NF-?B signaling pathway.(3)After stim?late the cells with m?ltiple antigens,then perform dual-luciferase reporter gene analysis,we found that NOD1 can intracell?lar recognize the stim?lation of iE-DAP.Therefore,it was hypothesized that NOD1 can recognize the pathogenic component of gram-negative bacteria in the cell,and then activates downstream NF-?B signaling pathway to nd induces immune responses.(4)The above spec?lations were verified by using qRT-PCR.The res?lts showed that after iE-DAP stim?lated,the NOD1 expression in cells was rapidly increased,and then activate NF-?B signaling pathway to induce the expression of downstream inflammatory cytokines.(5)Then,we found that after recognition of iE-DAP,NOD1 can recruits the downstream factor RIP2 to transmit the signal.Then we have studied the function of domains of NOD1,res?lt showed that the CARD domain was used to interact with RIP2,and the LRRs domain was used to recognize i E-DAP.