Cloning,Identification And Functional Analysis Of CD226 Subfamily Genes And Ligands Necl5 In Nile Tilapia(Oreochromis Niloticus)

In mammals,CD226 and CD96 are named as CD226 subfamily,which play an important role in mediating immune recognition,cell adhesion and T cell activation.However,the molecular function of CD226 subfamily in fish has not been reported.In order to fully explore the function of CD226 subfamily genes and their ligands in fish,this study took tilapia,an important economic fish,as the research object.Based on the cloning and identification of CD226 subfamily molecules and their ligands,the research focuses on their expression,interaction,regulation of downstream effectors and signal pathways:(1)qPCR was used to detect the expression of CD226 and CD96 in healthy tissues,as well as the expression of CD226 and CD96 in tissues or cells after immune stimulation;(2)the interaction of CD226 and CD96 with the ligand Necl5 was studied by subcellular co-localization and yeast two hybrid system;(3)the effects of CD226 and CD96 recombinant proteins and antibodies on the proliferation of whole lymphocytes and the expression of downstream effectors in tilapia head kidney whole lymphocytes were studied;(4)double luciferase reporter assay was used to detect the signal pathway they participated in.The results are as follows:1.Both CD226 and CD96 are involved in the immune response of tilapia against pathogensThe qPCR results showed that CD226 was mainly expressed in the brain of healthy tilapia,while CD96 was mainly expressed in the thymus.After stimulated by Streptococcus agalactiae and poly I:C,the expression of OnCD226 was significantly upregulated in thymus,head kidney and spleen,with the highest expression in thymus at 6 h;The expression of OnCD96 was also significantly up-regulated in thymus,spleen and brain,with the highest expression in spleen at 24 h.In addition,after stimulation by Streptococcus agalactiae,poly I:C and LPS,the expression of OnCD226 and OnCD96 in tilapia head kidney lymphocytes was also significantly up-regulated,and most of them reached the peak at 96h.Both OnCD226 and OnCD96 were up-regulated after the classical T cell dependent antigen hemocyanin(KLH)immunization,and the upregulated expression time after the second immunization was earlier than that of the first immunization,but the intensity was not always stronger than that of the first immunization.The results showed that OnCD226 and OnCD96 were involved in the immune response of tilapia to the invasion of pathogenic microorganisms,and it was likely to mediate host defense against T-cell-dependent pathogen infection.2.CD226 and CD96 interact with the ligand Necl 5 respectivelySubcellular co-localization results showed that OnCD226 and OnCD96 colocalized with OnNecl5 on the membrane respectively,indicating that OnCD226 and OnCD96 had the structural basis for interaction with OnNecl5.Further verification by yeast two hybrid experiment showed that protein interaction between OnCD226 and OnNecl5,and OnCD96 and OnNecl5 occurred at yeast two hybrid level,suggesting that the interaction between CD226 and CD96 and their common ligand Necl5 also exists conservatively in fish and plays an important role.3.OnCD226 and OnCD96 participate in the induced expression of downstream effect factorsThe results of cell proliferation experiment showed that rabbit anti-OnCD226-ECD and rabbit anti-OnCD96-ECD could enhance the proliferation of tilapia head kidney whole lymphocytes,but OnCD226-ECD and OnCD96-ECD had no significant effect;However,OnCD226-ECD and OnCD96-ECD significantly affected the expression of inflammatory factors(pro-inflammatory factor TNF-α and anti-inflammatory factor IL10),cell differentiation related factors(Th1 cells:IL-12,Th 17 cells:IL-17,terg cells:Foxp3),cell surface marker molecules(TCR-β and CD8)and ligand Necl5.4.Both CD226 and CD96 significantly activated NF-κB pathway and inhibited IRSE pathwayDual luciferase reporter assay showed that OnCD226,OnCD96 and OnNecl5 effectively activated NF-κB pathway,and up-regulated the expression of IFN1 and IFN3.STAT1 signal transduction factor was also activated accordingly.Interestingly,IRSE,IFN activated response element,was inhibited.These results indicate that CD226 subfamily may regulate the immune response of fish through NF-κB and interferon.