Preparation Of Monoclonal Antibodies Against Porcine IFN-β、γ And Establishment Of Porcine IFN-β Sandwich Elisa

According to the different immune functions, immune system can be divided into two parts:innate immunity and adaptive immunity. The innate system is the first line to defend infection by microorganisms such as virus, bacteria, it also contribute to the induction of adaptive immune responses. The secretion and expression of type Ⅰ interferon is an important part of innate immunity, its ininclude IFN-α and IFN-β. There are only three type cells can secret type Ⅱ interferon they are NK cell, CD8+T cell and CD8+T cell Th subtype, it ininclude IFN-γ, Type Ⅱ interferon is the major macrophage activating factor and also known as immune interferon. We have established a double antibody sandwich ELISA method for detection porcine IFN-β.The work in detail is as following:1. The cloning and expression of porcine IFN-β and IFN-γ geneAccording to the mRNA sequence on Gene Bank about porcine IFN-Pand IFN-y genes, two pairs of specific primers were designed. IFN-β and IFN-y were amplified by RT-PCR from the total RNA of PK-15cells.The two genes were all cloned into prokaryotic expression vector PET-28a and eukaryotic expression vector PCAGGS-HA and sequencing result showed that the cloned sequence were498bp and444bp in length. The prokaryotic expression vector of IFN-β, IFN-y were transformed into E.coli BL21and induced by IPTG, Inclusion Bodies and soluble protin were expressed. Then the eukaryotic expression vector of IFN-β, IFN-y were transfection into Hela cells and verified by Western Blotting.2. The preparation of monoclonal antibody against porcine IFN-βand IFN-γBalb/c mouse were immunized by purified prokaryotic expression product porcine IFN-β and IFN-γ respectively, when the serum titer reach to1:6400after the second immune after the second immune, qualified splenocytes were fused with SP2/0cell. After indirect ELISA screening and three cycles subcloning, we have obtained four McAbs against the IFN-β and eight against IFN-γ. Western Blotting and IFA confirmed that all these McAbs reacted specifically with eukaryotically expressed IFN-β and IFN-γ.3. The purification and enzyme-labeled of monoclonal antibody against porcine IFN-βFour strains hybridoma cells were expanded for production of ascites in a large quantity. Purified four strains monoclonal antibody by caprylic acid-ammonium sulfate precipitation (CA-CA) and verified after purification. The consistency of these McAbs reach to:2D126.9mg/ml、4A64.2mg/ml、2H63.4mg/ml、4G72.5mg/ml. The four McAbs were enzyme-labeled by HRP and the ELISA titers reached to:2D121:12800,4A61:12800,2H61:3200,4G71:1000.4. Establishment of porcine IFN-β sandwich ELISABy using CHO cell expressed IFN-β as antigen, a sandwich ELISA was developed using the2D12as capture antibody and4A6as detection antibody. Based on the optimization assay, the concentration of capture antibody was2μg/ml, the dilution of detection antibody was1:2000. PAM cells was stimulated by PRV and sampled at3h、6h、9h and12h after stimulation. IFN-β level in the medium was detected by the established ELISA methods. The results show that IFN-β level gradually improved with the stimulation time increasing, and the results consistent with previous study.