Generation Of Monoclonal Antibody Against PRRSV Nsp1α And Proteomics Investigation

Porcine reproductive and respiratory syndrome virus(PRRSV) is one of the most important pathogens affecting the swine industry worldwide. PRRSV can interfere the expression of type I interferons(I-IFN) in host cells and the signaling pathway mediated by I-IFN, thereby weak the immune response in infected pig cells. The PRRSV nonstructural protein 1α(nsp1α) inhibits the activity of I-IFN and plays crucial roles in the immune response of host cells infected by PRRSV. In addition, nsp1α plays crucial roles in the synthesis of viral subgenomic mRNAs, but the exact mechanism remains unclear. We prepared PRRSV nsp1α monoclonal antibodies in this study, and linked nsp1α gene to lentivirus vectors, then expressed in porcine alveolar macrophage cell lines PAM-3D4 cells(PAM-3D4). We analysed the extracted cellular proteins using iTRAQ quantitative proteomics technique. A total of 299 differentially expressed proteins which more than 1.2 times were identified. We also discussed the functions of PRRSV nsp1α in regulating the host innate immune response in this article. The main researches are as follows: 1. The prokaryotic expression and purification of PRRSV nsp1α proteinWe inserted nsp1α gene of PRRSV WuH3 strain into prokaryotic expression vector pET-28 a and constructed the prokaryotic expression plasmid pET-28a-nsp1α. Then the pET28a-nsp1α plasmid was transformed into E.coli BL21. The SDS-PAGE confirmed that nsp1α protein was highly expressed in E. coli which induced by IPTG and the nsp1α protein mainly exited in the form of insoluble inclusion bodies. We obtained the nsp1α protein with the concentration of 0.64 mg/mL after the insoluble inclusion bodies were purified. 2. The preparation of PRRSV nsp1α monoclonal antibodies and analysis of subtypesThe BALB/c mice were immunized by the purified nsp1α prokaryotic protein. Then we fused the immune spleen cells with myeloma cells and obtained 11 cell lines which could secret nsp1α antibodies sustainably after ELISA detected and three subcloning screened. They were named respectively as: 1A7, 1A10, 1C3, 2D3, 2E5, 2E7, 3B9, 3E4, 3E11, 3G2, 4E5. There are seven nsp1α antibodies(2E5, 2E7, 3B9, 3E4, 3E11, 3G2, 4E5) which have the specific interaction with nsp1α protein after confirmed by immunofluorescence(IFA) and Western Blot experiments. The subtypes result showed that all the light chains of nsp1α monoclonal antibodies are kappa chains; the heavy chains of six nsp1α monoclonal antibodies(2E5, 2E7, 3B9, 3E4, 3E11, 4E5) are IgG1 type, only the heavy chain of 3G2 monoclonal antibody is IgG2 a type. 3. The packaging and verification of lentivirus expressing PRRSV nsp1αIn order to study the effect of nsp1α on the protein expression of PAM-3D4 cells, the expression plasmid containing the nsp1α and lentiviral packaging helper plasmids were co-transfected into HEK-293 T cells. Then we collected the supernatant at 24 h and 48 h after transfection and verified via Western Blot and IFA, the results showed that the expression of the target gene could be detected and the best expression time of the purpose gene was determined at 72 h post-infection. 4. The analysis of PRRSV nsp1α proteomics dataThe lentivirus which expressing nsp1α infected PAM-3D4 cells and the cell samples were collected at 72 h, then the PAM-3D4 cell protein extract was analyzed by iTRAQ quantitative proteomics technique. There are 5126 proteins and a total of 299 differentially expressed cellular proteins which more than 1.2 times were identified, including 113 up-regulated proteins and 186 down-regulated proteins. These differentially expressed cellular proteins were mainly involved in cellular growth and proliferation, the innate immune response, and signal transduction. The UniProt database and ingenuity pathways analysis(IPA) were used to analyze the subcellular localization, functional characterizations and networks of the differentially expressed cellular proteins. Finally, we discussed and analyzed the regulation of nsp1α to host proteins in viral replication, inflammatory responses and apoptosis.