The Interaction Of Notch1a Molecule With Immune Gene Promoter In Zebrafish

The Notch signaling pathway plays an important role in cell-cell communication,especially in the terms of development and differentiation.But its function in innate immunity is still ambiguous,and whether these Notch receptors are involved in innate immune regulation and the mechanism of how to exert immune regulation is not clear.Innate immunity is a semi-specific,widely distributed immunity form,is the first line of defense against pathogens and a natural defense function that organisms gradually form during long-term evolution.Its recognition mechanism is mainly caused by pattern recognition receptors(PRRs)on the cell surface and cytoplasm and pathogen-associated molecular patterns(PAMP)of bacteria,viruses and fungi,and damage-associated molecular patterns(DAMPs).As the advantages of easy feeding,reproduction ability,rapid and in vitro development and transparent embryo,especially only possese innate immunity,zebrafish has been the organism that utilized in many different research fields.With the completion of the whole genome sequencing of zebrafish,its biological research has entered the era of verification of gene function and regulatory mechanism.Promoters,as switches in gene expression,play an important role in the expression pattern of genes.As gene function research continues to deepen,reporter gene technology is widely used to detect transduction of intracellular signaling pathways and monitor gene expression.This system can provide some complementary and supportive means for the study of regulation mechanism of antibody-deficient zebrafish protein levels.In order to study the effect of zebrafish Notch1 a on immune-related genes,this study used zebrafish as a model organism to identify the promoter of c3 a.1,c9,il-1?,il-6,il-10,il-11 b,il-12 a,inos2b,traf6,ticam1,myd88 genes,then the promoter region prediction,the location of CpG islands analyssion,and the TBP,NF-?B and others transcription factor binding sites were analyzed by bioinformatics tools.The primer pair was then designed based on promoter for cloning.A reporter gene recombinant plasmids were constructed and its activity was verified.To examine whether there were differences in promoter activity in different cells,we transfected successfully constructed promoter reporter genes were into HEK293T,RAW264.7,EPC,GCO and ZF4 cell lines respectively.And then we stimulated transfected cells with different TLRs ligands to determine which one is suitable for immunology research.Finally,follow-up stress experiments,the zebrafish N1 a ICD recombinant plasmid and c3 a.1,c9,il-1?,il-6,il-10,il-12 a,inos2b,traf6,ticam1,myd88,il-11 b,tnf?,ifng-1-1,rela was co-transfected into HEK293 T and RAW264.7 to verify the regulation of N1 a ICD on each molecule under resting and stress state.The experimental results obtained were as follows:(1)Have obtained the promoter sequences of c3 a.1,c9,il-1?,il-6,il-10,il-11 b,il-12 a,inos2b,traf6,ticam1,myd88 gene by genome BLAST and analysed the sequence by bioinformatics methods.Results indicated that the promoter region and TSS,TBP and NF-?B binding sites were present in the obtained promoter sequences,but the GC content was low.The potential transcription factor binding sites on the promoter sequence mainly include transcription factor binding of various families such as POU family,Forkhead family,Nkx family,GATA family,Hox family,IRF family,NF-?B family,RA family and Sox family.It also includes key molecules such as AP-1,CREB,Evi,C/EBP,TBP and Sp1.(2)Have amplified the target fragment by PCR and recomnination with pGL3-Enhancer reporter vector and the constructed immunogenic promoters' activity was examined in HEK293 T.Different fragment length promoters of il-11 b were tested in HEK293 T cells.The cloned promoters are all active,and the core region of the il-11 b promoter may be present between-2000 bp and-1000 bp.The cloned promoter can be used for subsequent studies.(3)The constructed traf6 promoter report gene was transfected into HEK293 T,RAW264.7,EPC,GCO and ZF4 to test the promoter activity in different cell lines and they impacted the promoter activity significantly,EPC and GCO activity being lower,RAW264.7 followed,and ZF4 and HEK293 T higher.However,after TLRs ligands stimulation treatment,HEK293 T showed no changes,and the other four cell lines all had stress response.Combining various factors,HEK293 T can be used to verify the regulation of immune-related genes by Notch molecule at rest,and RAW264.7 is suitable for the regulation of immune genes by Notch molecule under TLRs ligands stimulation.(4)Notch1a intracellular domain and reporter gene were co-transfected into HEK293 T.At rest,N1 a ICD inhibited the promoter activities of c3 a.1,il-1?,il-6,il-12 a,inos2b,traf6,ticam1,and myd88 while enhanced the activity of il-10 and c9 promoter activity.This indicates that the Notch molecule inhibits the transcription activity of proinflammatory complement,cytokines and transfer protein at rest.In RAW264.7,under the stimulation of TLRs ligands,N1 a ICD exerted a very strong inhibitory effect on il-1?,il-10,tnf? and ifn?,and also strongly inhibited traf6,myd88,ticam1 and rela.This indicates that Notch participates in immune responses through multiple immune signaling pathways under TLRs ligands stimulation.Overall,this study used bioinformatics methods and dual luciferase reporter gene technology to clone and validate key molecules in the zebrafish complement system,related molecules in cytokines,and key transport protein promoters in immune signaling pathways.And preliminary study on the regulation of immune-related signals by zebrafish N1 a ICD at rest and stimulation were carried out.