Generation Of Monoclonal Antibody Against PRRSV Nsp1β And Proteomics Investigation

In 1987, Porcine Reproductive and Respiratory Syndrome(PRRS) was firstly discovered in the United States, and it has been also found in Europe, which causing huge economic losses to the pig industry in the world. PRRSV is the pathogenic factor of PRRS. PRRS is a pig virulent infectious disease. Studies have shown that some virus incoulding PRRSV could inhibit the production of interferon and escape immune surveillance. Thus, they could inhibit the production of type I interferon which largely attributing to nonstructural protein by blocking the activition of the promoter of IFN-β and NF-κB. PRRSV nsp1β plays an important role in the regulation of host immune responses, however, the precise mechanism remains to be further studied. In view of the current studies of nsp1β, we study the biological function of nsp1β from the perspective of proteomics. In this study, we successfully preparated the PRRSV nsp1β monoclonal antibody and obtained the PRRSV nsp1β recombinant lentivirus. Then we identified 425 differentially expressed proteins in porcine alveolar macrophage cells PAM 3D4/21 infected by PRRSV nsp1β recombinant lentivirus compared with controls using iTRAQ coupled with LC-MS/MS proteomics technology, and these proteins are involved in innate immunity, apoptosis and other biological process. The main research contents are as follows:1. Expression and purification of PRRSV nsp1β The PRRSV nsp1β was successfully connected to the p GEX-6p-1 vector. Then we used the prokaryotic expression system E.coli BL21(DE3) to express recombinant protein. We explored different inducing expression conditions, and the optimal recombinant protein expression of condition was determined that E.coli BL21(DE3) was induced by 0.8 m M IPTG 5h at 37℃, the protein exists in the form of inclusion body in the E.coli BL21(DE3). After dialysis, the purified protein was obtained with the concentration of 1.5mg/m L.2. Preparation of monoclonal antibody against PRRSV nsp1β The purified nsp1β protein was used as the immunogen, and the six week old female BALB/C mice were immunized with the purified protein. Then we obtained 3 strains with stable hybridoma cell lines after cell fusion, ELISA experiment and cell clone, and they were named as 1G7, 2A8, 4G10. The 3 cell lines were detected by IFA and Western Blot, and the results shows that all of which could interact with PRRSV nsp1β specifically. We also identified the subtypes of PRRSV nsp1β monoclonal antibodies, and the heavy chains are all IgG1 type, light chains are all κ chain.3. Packaging of PRRSV nsp1β recombinant lentivirus In this study, we used the lentivirus package system inclouding four plasmids, PLP1, PLP2, VSVG and CMV-MCS-3flag-EF1-cop GFP-T2A-Puro(the lentivirus expression vector) to package the nsp1β recombinant lentivirus. The four plasmids were co-transfected into HEK-293 T cells. PRRSV nsp1β was constructed into the host cell subgenomic system, and the expression of nsp1β was verified by IFA experiment.4. Analysis of PRRSV nsp1β proteomics data In order to further study the role played by nsp1β, we investigated the biological function of PRRSV nsp1β through the proteomics in this study. We detected 425 differentially expressed proteins in nsp1β-overexpressed PAM 3D4/21 cells through iTRAQ technique, including 186 up-regulated and 239 down-regulated proteins. Bioinformatics analysis was used to analyze the subcellular localization, biological function and protein interaction network of differential protein. Differentially expressed proteins identified in this study are related with energy metabolism, protein expression, apoptosis, immune and so on. This suggests that PRRSV nsp1β could contribute to the evasion from the host immune surveillance and the replication of PRRSV. This study also explains the role of nsp1β in the regulation of host cell proteins during viral infection.