Immunological And Proteomics Analysis Of Mouse Bone Marrow Dendritic Cells Infected ORFV

Orf,also known as Contagious Pustular Dermatitis of Sheep,is an acutely contagious zoonosis caused by ORF Virus?ORFV?in sheep,goat etc.Orf virus?ORFV?is recognized as a strong host immune function.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.Dendritic cells?DCs?are the most powerful professional antigen presenting cells?APCs?in the body,and play an important role in the regulation of innate immunity and adaptive immunity.However,it is unclear how DC exerts its immunoregulatory effect after being infected by ORFV.In this experiment,ORFV and mouse bone marrow DCs were used as targets and it was determined by indirect immunofluorescence that ORFV can infect DCs based on the successful preparation of ORFV129 gene polyclonal antibodies.The infection of ORFV significantly promotes differentiation and maturation of DCs by detecting surface marker.Finally,the differential proteomes of DCs infected by ORFV were analyzed.The implementation of this study provides a platform for the successful isolation of mouse bone marrow DCs,and lays a foundation for further revealing the immune mechanism of ORFV-infected hosts.The conclusions are as follows:1.A potency of 1:3200 rabbit serum was obtained.2.Higher purity of DCs were successfully prepared and it was can promote the maturation of DCs when stimulated by ORFV:Balb/c bone marrow DCs were aseptically isolated and cultured.Cytokine-induced mouse bone marrow cells shows obvious burrs after 6 days.The expression rates of cell surface molecules MHC-II,CD86,and CD40 in ORFV(10^-1)-stimulated groups were increased compared to the control groups.The expression of IL-12,IL-1?,TNF-?,IL-6,INF-?cytokines were significantly increased and IL-10 was decreased as compared to control groups.The uptaking FITC-dextran ability of DCs in ORFV(10^-1)-stimulated group were decreased.At the same time,the proliferation ability of T-lymphocytes at different concentration co-cultured with DCs were significantly higher than untreated group.3.By using of iTRAQ technology combined with LC-MS/MS mass spectrometry,Identification of differentially expressed proteins were analyzed.A total of 191 types of differential proteins were identified,134 of which were up-regulated?such as Scin?,and others were down-regulated?Npl,Cstb etc.?.biological processes-based identified the differentially expressed proteins that were mainly involved in antigen processing and presentation,cell biosynthesis,translation,immune response,and defense against external stimuli.KEGG pathway analiyais showed that these differentially expressed proteins taked particate pathway,Antigen processing and presentation,ribosome synthesis pathway,Cytosolic DNA-sensing pathway,Glutathione,cysteine and methionine metabolism and the course of some diseases.4.WB test using anti-Scin,Npl and Cstb protein anti-bodies and anti-Mouse IgG,HRP Conjugate,respectively.The result shows that is up-regulated and two down-regulated proteins are consistent with the results obtained by the iTRAQ proteomics technique.