Screening Differentially Expressed Proteins In The BPIV3-infected MDBK Cells And Those Reaction For Infecting Host Cells

The bovine parainfluenza type 3,belongs to the member of family Paramyxoviridae,Respirovirus genera,is an enveloped,single-strand negative-sense RNA virus.It has been reported that BPIV3 infection not only recruits host cell proteins to participate in viral replication,but also activates cellular related signaling molecules to help fulfillment of its lifecycle.Therefore,it is important to analyze the host cell proteins involved into BPIV3 replication,and further investigate the changes of signaling pathway during BPIV3 infection.Thus in this study,i TRAQ proteomics technology was used to analyze the host cell proteins participating into the BPIV3 replication,and the differentially expressed proteins were obtained in the BPIV3-infected host cells.The main results were as follows:1.Generation of Mc Abs specific for BPIV3 and biological characteristic analysisThree positive Mc Abs specific for BPIV3 were generated by using BPIV3 as immunogen.The Mc Abs had the good reactivity with BPIV3,providing a basis for subsequent experimental detection.On the basis of Mc Abs production,DAS-ELISA detection method was established.The DAS-ELISA has good specificity,sensitivity and repeatability.The DAS-ELISA is suitable for detection of veterinary basic clinical samples.2.Proteomics of BPIV3 infected MDBK cellsTo explore the protein expression of BPIV3 infected the host cell,the protein expression in BPIV3 infected MDBK cells were tested and analyzed by used i TRAQ combined with the high-throughput proteomics technology 2D LC-MS/MS.A total of 116 differentially expressed proteins were identified,74 protein expression increased and 42 protein expression down-regulated.The bioinformatics technology analysis showed that the differential proteins were mainly matched in signal transduction,lipid metabolism,infectious disease,immune response and inflammatory response.The expression of 8 differentially proteins were detected by q RT-PCR method to verify the reliability of proteomics results.The results of q RT-PCR were consistent with that of proteomics analysis.3.The activation of the p38 MAPK signaling pathway by BPIV3 infectionIn the previous proteomics studies,the level of MKK3 recognized as the upstream kinase of p38 in BPIV3 infection group was significantly increased compared with the control group.Therefore,the role of p38 MAPK pathway in the process of BPIV3 infection was studied.Firstly,the results showed that the activation of MKK3,which had the benefits of the viral multiplication,and phosphorylation of p38 were induced after BPIV3 infected.p38 MAPK signaling pathway participates in the replication of BPIV3.The level of IL-6,IL-8,IL-13 and TNF-α were tested by the ELISA,which proved that the p38 MAPK signaling pathway participates in the inflammatory response induced by BPIV3.5.The role of cholesterol in MDBK cells infected by BPIV3In the results of previous proteomics studies,there were many proteins in the process of lipid metabolism.LDLr and SAR1 A are key proteins to regulate endocytosis and outside of cholesterol.To investigate the role of cholesterol in BPIV3 infection,the MβCD was employed to remove the cell membrane cholesterol and the viral envelope cholesterol.The results suggested that the cell membrane cholesterol was indipensible for BPIV3 entrance into cell and its replication.Removing of viral envelope cholesterol inhibited the infectivity of BPIV3.Above the results showed that expression proteins in MDBK cells changed between BPIV3 infection group and control group.The differential expression proteins associated with BPIV3 infection were identified by the expression profile analysis.Their functional analysis proved that the p38 MAPK and cholesterol molecules played an important role in the BPIV3 infection.All the studies provide the theoretical foundation to reveal the pathogenesis mechanism.