Differences In Immune Response And Transcriptional Levels Of Murine DCs Induced By ORFv And CBP Proteins

Contagious Ecthyma virus of sheep,also known as Orf virus(ORFV),can cause acute,contagious skin infectious diseases in goats and sheep.The diseased sheep are mainly manifested in the formation of erythema,papules,pustules,ulcers and sputum thick sputum in the lips and oral mucosa.ORFV can cause a strong immune response after infection and ORFV immunomodulatory effects on the host regulatory by encoding a large number of protein to achieve immune or inflammatory response in the host immune pathway.CBP protein is an important virulence proteins of ORFV expressed in early,which play an important role in regulating host immune responses.DC is the most important antigen presenting cell(APC)and is the only APC in the body that can directly stimulate the proliferation of primary T cells.DCs play an important role in immune response of immune regulation and mediate peripheral immune tolerance.However,there are few studies on how ORFV and its virulence protein CBP interact with mouse bone marrow-derived DCs in vitro.This experiment was carried out by preparing prokaryotic protein CBP,and then detecting whether ORFV and protein CBP promoted differentiation and maturation of DCs cells by surface markers,cytokines and phagocytosis experiments.Finally,the differential genes were analyzed.This study lays a theoretical foundation for the interaction between ORFV and host immune cells,and facilitates further elucidation of the immune mechanism of ORFV-infected hosts.The conclusions reached are as follows: 1.The prokaryotic expression vector pET-28a(+)-ORFV112 was constructed and the protein was successfully expressed and purified.2.Higher purity of DCs were successfully prepared.Three different concentrations of ORFV can significantly promote the expression of co-stimulatory molecules CD86 and CD40 on DC surface,and also promote the secretion of cytokines such as IL-1β,IL-6,IL-12 and TNF-α.Viruses at concentrations of ORFV(10-1)and ORFV(10-2)significantly inhibited the expression of the surface costimulatory molecule CD80.The costimulatory molecules and secreted cytokines of DC also increased significantly,similar to the effect produced by the virus after DC.In summary,ORFV and 100 ng CBP protein can induce the maturation of mouse DCs and induce T cells to differentiate into Th1 cells,and promote the release of IFN-γ cytokines by T cells to increase the body’s effect on external stimuli.3.Identification of DC differentially expressed genes after ORFV and protein treatment by RNA-seq sequencing.It was found that there were 8241 differential genes,4205 up-regulated genes and 4036 down-regulated genes in ORFV-stimulated DCs compared with unstimulated cells.After CBP stimulated DC,there were 7301 differential genes,3535 up-regulated genes,and 3766 down-regulated genes compared with unstimulated cells.By analyzing these differential genes,differential genes with common changes are concentrated in cytokine-cytokine receptor interaction signaling pathways,antigen presentation and processing,T cell receptor signaling pathways,and cell cycle and apoptosis.There is a certain difference in the changes caused by the transcriptional level of the sheep aphthous virus and the CBP protein.4.The CD8 B,Ctse,IL2 RA,Atp5e,CD80,Lck,and CCL8 genes were significantly up-regulated compared with the DC group after virus and protein treatment,while the genes Cstb,Akt,and CCR1 were significantly down-regulated compared with the DC group.RNA-seq sequencing results were consistent.