Effects Of Peste Des Petits Ruminants Virus And Its N Protein On The Immune Response Of Peripheral Blood Mononuclear Cells Of Goat

Peste des petits ruminants virus(PPRV)infection can cause an acute,fever,and highly contagious diseases in small ruminants(goats or sheep).Goats are more susceptible to PPRV infection,and sick goats mainly show fever,increased secretions in eye and nose,pneumonia and diarrhea,and other symptoms.Studies have shown that PPRV infection in goats can induce the immune suppression response in animal body,leading to produce inflammatory response.N protein is the immunogenic protein with the highest expression level of PPRV during infection and plays an important role in the immune response of PPRV infection.PBMCs are kinds of immune cells,including lymphocytes(T cells,B cells),monocytes and dendritic cells.PBMCs play an important role in immune regulation and pathogen recognition,and can induce early innate immune response of host defense.At present,there are few studies on the interaction of PPRV and N proteins with goat PBMCs in vitro.Therefore,in this study,prokaryotic expression and purification of PPRV N protein were firstly performed,and rabbit polyclonal antibodies against N protein were prepared by using recombinant N protein as antigen.Then PPRV and N protein were used to stimulate the PBMCs,and the results confirmed that PPRV can proliferate and replicate in PBMCs.CCK-8,qRT-PCR,ELISA,flow cytometry,techniques were also explored to examine the production of cytokines,the expression of surface markers,cell proliferation and NO release and evaluate the changes of immune response in PPRV-infected or N protein-treated PBMCs.This study provides a theoretical basis for studying the interaction between PPRV and host cells,and also lays a foundation for the immune response and pathogenic mechanism of PPRV-infected hosts.Through the above contents,the following conclusions are obtained.1.The recombinant N protein of PPRV was successfully constructed,expressed,purified,and the high-potency polyclonal antibody of N protein was prepared.2.High purity PBMCs of goats were successfully isolated.PPRV could infect PBMCs in vitro and proliferated and replicated in cells.PPRV infection significantly inhibited the proliferation of PBMCs.PPRV infection also induced the expression of IL-10,1L-6,EL-10,IFN-? and TNF-? mRNA,as well as the secretion of cytokines such as IL-1?,IL-6,IL-10,IFN-? and TNF-? in the cell culture supernatants.It could also promote the expression of CD4,CD8,CD40,CD86 and CD80 of PBMCs and participate in the cellular immune response.3.In addition,PPRV infection also enhanced the release of NO,which is closely related to cytokines and inflammatory responses,and inflammatory cytokines mediated the production of NO.4.Recombinant N protein of PPRV had an obvious inhibitory effect on the proliferation of PBMCs,It also increases the release of NO.N protein treatment induced the mRNA expression of IL-6,IL-10,TNF-?,and IFN-y and promoted the secretion of cytokines in PBMCs,which is related to the role of N protein invirus replication.N protein treatment also promoted the expression of surface markers CD4,CD8,CD40,CD86 and CD80 to participate in the immune response of the hosts.The above research results lay a foundation for the understanding of the interaction between PPRV and N proteins and host cells and the immune effect.It provides a new clue for the prevention and treatment of PPRV and the development of vaccine.