| Porcine reproductive and respiratory syndrome(PRRS)is a highly contagious disease that seriously harms the global pig industry.The disease is characterized by high fever and high mortality,resulting in miscarriage and stillbirth in the middle and third trimester of pregnant sows,death of piglets and slow growth of fattening pigs.The disease has caused huge economic losses to the worldwide pig industry in the past two decades.The pathogen of PRRS is porcine reproductive and respiratory syndrome virus(PRRSV).The existing PRRSV prevention and control methods mainly rely on vaccine immunity.However,due to the continuous variation of PRRSV itself and the ability to escape the inherent and acquired immune response after infecting the host through a variety of mechanisms,the prevention and control of PRRS is still facing great challenges.The adaptive cellular immune response induced by PRRSV infection(killing T cell,CTL)appears and is at a low level 2 weeks after infection)may be related to the down-regulation of porcine MHC-I class molecule(porcine leukocyte antigen-PRRSV I)expression or antigen presentation function of porcine alveolar macrophage(PAMs)induced by PRRSV infection.Further studies have confirmed that a number of non-structural proteins encoded by PRRSV,such as NSP1α,NSP2TF and NSP4,are involved in the regulation of antigen processing and presentation mediated by MHC-I molecules.The specific antibodies induced by PRRSV appeared one week after infection,but the main recognition of non-structural protein(NSPs)and nucleocapsid protein(N),were non-neutralizing antibodies,but the mechanism was not clear.Dendritic cell(DCs)plays a key role in initiating and activating adaptive immune response and can be effectively infected by PRRSV.Previous studies have found that the transcriptional expression of porcine leukocyte antigen-II(SLA-II)is down-regulated while the protein expression is unchanged or up-regulated after PRRSV infection with DCs.At present,there is no in-depth study on whether the infection of DCs by PRRSV affects the MHC-II class processing and presentation of viral proteins mediated by PRRSV and whether the specific PRRSV antigen presented is related to the humoral immune response of PRRSV.Therefore,by studying the interaction mechanism between PRRSV and host antigen presenting cells(dendritic cell(DCs)),this study explores the process of immune system processing and presenting viral proteins,inducing the activation of CD4~+T cells and B cells,and revealing the causes of the body’s special humoral immune response to PRRSV.The work and results of this study are as follows:1.Preparation and activity detection of monoclonal antibodies against SLA-DRβThrough the construction of the vector,the target fragment of SLA-DRβamplified by PCR was connected to the p ET-28a expression vector,and the prokaryotic expression of SLA-DRβprotein was successfully expressed.The high purity SLA-DRβprotein was further purified by Ni column.By immunizing BALB/c mice,the titer of antibody in serum of mice reached more than 10~5,which reached the standard of monoclonal preparation.Through cell fusion technique and monoclonal screening,a monoclonal antibody which can react with SLA-DRβrecombinant protein was successfully obtained.The antibody was purified from ascites and the subtype was identified as Ig G1.The reaction with PAMs cell lysate and Western blotting(WB)showed that the monoclonal antibody could specifically bind to endogenous SLA-DRβ,which could be used for further research.2.Effect of RRSV infection on the expression of SLA-DR in DCs cellsFluorescent quantitative PCR(q PCR)and WB(self-made anti-SLA-DRβmonoclonal antibody)were used to detect the m RNA and protein expression of SLA-DRβin cells infected with BM-DCs at different time and different PRRSV strains,respectively.The results showed that the transcription level of SLA-DRβwas down-regulated and the protein expression level was up-regulated in BM-DCs at different times of PRRSV infection(8,12,18,24 h).Different strains of PRRSV could down-regulate the expression of SLA-DRβat transcriptional level.The protein expression level of SLA-DRβwas up-regulated in different strains.Flow cytometry was used to detect the expression level of SLA-DR on the cell surface of different PRRSV strains infected with BM-DCs 24 h.The results showed that the SLA-DR expression of BM-DCs was significantly up-regulated after PRRSV infection,suggesting that this universal upregulation of SLA-DR molecules may have a certain physiological function.That is,the presentation of PRRSV-NSPs by BM-DCs can induce the production of anti-PRRSV-NSPs antibodies in animals in the early stage of infection.3.Verification of the relationship between the presentation of PRRSV-NSPs-derived Antigen Peptide by SLA-DR on BM-DCs and the production of specific Antibody induced by itThrough the analysis technique of"SLA-DR-immune peptide library"established by our laboratory,we have obtained the information of high affinity immune peptide from PRRSV protein presented by SLA-DR on BM-DCs.In order to verify the above hypothesis,seven PRRSV-NSPs-derived antigenic peptides identified by mass spectrometry were selected and expressed artificially.The reactivity of PRRSV-NSPs-derived antigenic peptides with PRRSV-infected positive sera was determined by the"luciferase-linked antibody capture technique"and peptide ELISA detection technique established and optimized by the laboratory.The results showed that the results of LACA detection were consistent with those of peptide ELISA.There were antibodies specifically recognizing PRRSV-NSPs-derived antigenic peptides in the sera of pigs infected with PRRSV.To sum up,after PRRSV infection,the protein expression of SLA-DRβand SLA-DR on BM-DC was up-regulated,and specific antibodies were induced by PRRSV-NSPs-derived antigen peptides presented by SLA-DR molecules on DC.From the point of view of the processing and presentation of viral protein antigens after DC infection with PRRSV,this study revealed the molecular mechanism of a large number of PRRSV-NSPs recognition antibodies produced in the early stage of porcine infection with PRRSV. |
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