Applied Research Of Immune Regulation Funcation Of Duck CD40

Co-signal molecules are cell surface molecules that can transduce a signal to a cell to modulate an immune response positively(co-stimulate)or negatively(co-suppress).Costimulatory signals are key factors in determining whether T/B cells are capable of responding to specific antigens and ultimately mediating an appropriate immune response.In consideration of the fact that there is no data for duck co-stimulatory molecule CD40,so we cloned,verified and expressed it.To explore the role of duck CD40 in antiviral immune regulation.The main resultsare as follows:1 Molecular cloning,sequence analysis and verification of duck CD40The duck CD40 gene was successfully cloned by RT-PCR based on the design of primers for the avian CD40 gene sequence.The duck CD40 gene sequence contains a open reading frame(516 bp in length)encoding 171 amino acids.Multiple sequences alignments indicating that it show highest similarity to goose CD40,up to 87%,followed by chicken CD40 with a similarity of 81%.The similarity of duck CD40 and fish CD40 is higher than that of duck CD40 and mammalian CD40,its similarity to human CD40 is only33%.The predicted duck CD40 secondary structure shows that it containing a typical TNFR structure,and consist of extracellular regions,transmembrane regions,and intracellular regions.In order to further verify the molecule size of duck CD40,duck CD40 was expressed in E.coli prokaryotic expression system and purified for preparation of the mouse anti-duck CD40 polyclonal antibody.The peripheral blood lymphocytes of duck were isolated and cultured in vitro and the lipopolysaccharide was added to induce the expression of CD40,CD40 polyclonal antibody was used to detect the size of CD40 via western botting.The results showed that CD40 molecule was about 18 k Da,which was consistent with the expected protein molecular weight 18.64 k Da.2 Tissue expression profile of CD40 in ducklings and adult ducksRT-qPCR was used to detect the expression of duck CD40 in ducklings and adult ducks.The results showed that duck CD40 was widely expressed in ducklings and adult ducks’ tissues.The tissues with high expression level in ducklings were Harderian gland,pancreas,kidney,small intestine,lung,bursa,blood and spleen,and the expression of CD40 was lower in liver,thymus and muscle.In adult ducks,the tissues with higher expression was blood,Pancreas,heart,lung,kidney,Hastelloy and liver.And the expression of CD40 in muscle,lymph and muscular stomach was low.3 Study on antiviral immune regulation function of duck CD40The expression changes of duck CD40 and CD40 L in duck embryo fibroblast(DEF)cells infected with duck plague virus(DPV)and duck hepatitis a virus 1(DHAV-1)were detected by RT-qPCR.The results showed that DEF cells infected with DPV and DHAV-1,the expression levels of CD40 and CD40 L were significantly up-regulated(P<0.05 or P<0.01)at 24 hours post infection.At 48 h post infection,the expression levels of CD40 and CD40L were up-regulated on the basis of 24 h(P<0.05 or P<0.01).The recombinant CD40L(ligand of duck CD40)was expressed in E.coli prokaryotic expression system to obtain soluble duck CD40 L recombinant protein.DEFs were treated with exogenously CD40 L protein,to verify the activation of CD40 and its downstream signal pathway,the transcriptional expression of cytokines IL-6 and IL-8 were detected.The results indicate that IL-6 and IL-8 transcription levels were significantly up-regulated at 24 h post treated,up to 60-fold and 8-fold,respectively;at 36 h post treated,the upregulated levels of IL-6 and IL-8 were significantly lower than that of 24 h,up to10-fold and 1-fold,respectively.To investigate the effect of duck CD40/CD40 L ligation on viral replication,DEF cells were overexpressed duck CD40(A)containing 3 TNFR domains and duck CD40(Z)and CD40(G)with only 1 TNFR domain,and then treated with CD40 L proteins in infected with DPV,DHAV-1 and TMUV.RT-qPCR was used to detect viral copy number.The results showed that at 24 h post infection,there was no significant difference in viral copy number in all three groups infected with viruses compared with the control group,however,at 36 h post infection,the copy number and viral titers of TMUV in expressed CD40(A)experimental group showed a significant decrease compared to the control group,but there was no significant difference in CD40(Z)and CD40(G)group compared with control group,furthermore,there is no significant difference in DPV and DHAV-1 infected groups.In conclusion,duck CD40 is consist of 171 amino acids,with a typical TNFR structure,which is widely expressed in ducklings and adult duck tissues;DPV and DHAV-1 infection can up-regulate the expression of CD40 and its ligand CD40L;DEF cells treated with CD40 L protein resulted in upregulation of of CD40 and its downstream cytokines IL-6 and IL-8 in transcriptional level.DEF cells overexpressed duck CD40 containing three TNFR domains and then treated of CD40 L protein can control the viral replication of TMUV.