Study On The Targeting Effect Of Human Dendritic Cell Targeting Peptide On Porcine Dendritic Cells

Dendritic cells(DCs)are the most powerful professional antigen-presenting cells(APCs),which are present in the gastrointestinal tract widely.They are an important role inregulation immune responses,and connecting innate and adaptive immune system.Dendritic cell targeting peptide(DCpep)is a short peptide that can specifically recognize the ligands on the surface of DCs,and has powerful antigen presentation ability.Human-derived dendritic cell targeting peptide(DCpep)was successfully screened,and it is currently found that human-derived DCpep can specifically bind to DCs of chimpanzees,poultry,dogs,horses and cats and oral vaccines expressing human-derived DCpep can elicit higher mucosal immunity and specific immune response,however,there is no experimental study on whether human-derived DCpep can specific recognition of porcine-derived DCs.Therefore,exploring whether human-derived DCpep targets porcine-derived DCs and how human-derived DCpep affects the immune function of porcine-derived DCs is of great significance for the development of new targeted oral vaccines for porcine.In this experiment,density gradient centrifugation was used to isolate peripheral blood mononuclear cells(PBMCs)from the peripheral blood of piglets aged 5-8 weeks.and the immature monocyte derived dendritic cells(Mo DCs)were obtain after isolation and induction culture.The results of morphological observation,molecular phenotype and phagocytic function test showed that the method we used can obtain high purity Mo-DCs.In order to determine whether human DCs targeting peptide(DCpep)can be targeted to porcine DCs,we incubated the synthesized human DCs targeting peptide(DCpep-FITC)with porcine DCs,and detected of the binding of DCpep-FITC to porcine DCs by fluorescence microscopy and flow cytometry.The results showed that human DCpep could specifically bind to porcine DCs.To explore the mechanism of DCpep enhancing intestinal mucosal immunity induced by recombinant Lactobacillus.The recombinant lactobacilli(p PG-T7g10-ps420-DCpep/L.casei 393)fusion expressing DCpep and PEDV antigen epitope ps420 were used as the research objects and the recombinant Lactobacillus p PG-T7g10-ps420/L.casei 393)expressing antigen epitope ps420 was used as the control.After the above recombinant lactobacilli were incubated with Mo DCs,the ability of the Mo DCs to recognize and phagocytize the recombinant Lactobacillus were observed by scanning electron microscope.The results showed that the Mo DCs could recognize and phagocytize p PG-T7g10-ps420/L.casei 393 and p PG-T7g10-ps420-DCpep/L.casei 393.But,the number of p PG-T7g10-ps420-DCpep/L.casei 393 captured by porcine Mo DCs was higher than p PG-T7g10-ps420/L.casei 393.In order to determine the effect of dendritic cell targeting peptide on the activation of Mo DCs by recombinant Lactobacillus.In this study,the recombinant Lactobacillus p PG-T7g10-PPT/L.casei 393?p PG-T7g10-ps420/L.casei 393 and p PG-T7g10-ps420-DCpep/L.casei 393 were co-cultured with porcine Mo DCs,then,porcine Mo DCs were harvested at 6 h,12 h,and 24 h after co-culture.The expression of surface molecules and related cytokines of the porcine Mo DCs were detected by flow cytometry and q RT-PCR.The results of flow cytometry showed that the expression of MHC-II and CD80/86 on the surface of Mo DCs were increased by recombinant Lactobacillus p PG-T7g10-ps420/L.casei 393 and p PG-T7g10-ps420-DCpep/L.casei 393.And the more effective contribution of the recombinant Lactobacillus p PG-T7g10-ps420-DCpep/L.casei 393 was detected.The result of q RT-PCR showed that the expression of TLR-2,4,6,9 and IL-12,IFN-?,IL-10 were not significantly affected by recombinant Lactobacillus p PG-T7g10-PPT/L.casei 393,however,the m RNA expression of TLRs and related cytokines on the surface of Mo DCs was significantly increased by recombinant Lactobacillus p PG-T7g10-ps420/L.casei 393 and p PG-T7g10-ps420-DCpep/L.casei 393,and the effect of recombinant Lactobacillus p PG-T7g10-ps420-DCpep/L.casei393 was stronger than that of p PG-T7g10-ps420/L.casei 393.To verify the effect of DCpep on the ability of recombinant Lactobacillus to promote the proliferation of T cells stimulated by porcine Mo DCs,the Mixed Leucocyte Reaction was performed in this study.Use unstimulated Mo DC as a control group,Used Mo DCs stimulated with the recombinant Lactobacillus p PG-T7g10-PPT/L.casei 393?p PG-T7g10-ps420/L.casei 393 and p PG-T7g10-ps420-DCpep/L.casei 393 as stimulator cells,Use CD4~+T cells as responder cells,and CCK-8 kits to detect the proliferation of CD4~+T cells.Experimental results show that the mature Mo DCs stimulated by recombinant Lactobacillus can induce CD4~+T proliferation,and the effect of recombinant Lactobacillus p PG-T7g10-ps420-DCpep/L.casei 393 was stronger than that of p PG-T7g10-ps420/L.casei 393.The supernatant in the co-culture system of CD4~+T cells and Mo DCs was examined by ELISA,the results showed that the secretion of IFN-?,IL-12,IL-10 and IL-17was increased,and the effect of recombinant Lactobacillus p PG-T7g10-ps420-DCpep/L.casei 393was stronger than p PG-T7g10-ps420/L.casei 393.In conclusion,human-derived DCpep can be combined with porcine-derived DCs specifically,which is beneficial for human-derived DCs to capture recombinant Lactobacillusand stimulate the maturation of porcine-derived Mo DCs.In addition,human-derived DCpep can promote the antigen presentation of DCs and the proliferation of T cells,so as to improve the efficiency of recombinant Lactobacillus to deliver the antigen to DC.The results of this study provide a theoretical basis for the development of porcine DCs targeted oral vaccines.