Preparetation And Identificaton Of A Strain Of Hybridoma Cell Producing The Monoclonal Antibody Against Porcine Interferon Gamma

Interferon (IFN)-γis a kind of cytokines first originally discovered and mostly implied. It have not only an effect on anti-virus and anti-tumor, but also an enhancement of MHCIIantigen expression and efficient up-regulation of the antigen presenting pathway. This study was aimed to establish indirect ELISA for recombinant porcine IFN-γdetection, research the biological function and the mechanism of the porcine interferon-gamma (PoIFN-γ) in immune response, and provide a new method for diagnosis of porcine infectious disease.In present study this gene was expressed and purified, then generated a monoclonal antibody (mAb) preparated and identified.The recombinant PET-32-PoIFN-γand PGEX-4T-1-PoIFN-γwere cultured respectively, induced to express by IPTG. And the molecular weight of the fusion protein was about 43.0 and 35.0 KD as we expected. In SDS-PAGE, the molecular weight of His-PoIFN-γwas 35.0 KD, the expressed protein mainly located in inclusion bodies. Recombinant protein was harvested by centrifugation and disturbed by sonication, and then inclution body was extracted. The protein used as immune antigen. The molecular weight of GST-PoIFN-γwas 43.0 KD.And the molecular weight of GST was 26.0 KD. The recombinant protein was purified by GSTrap FF affinity chromatography. SDS-PAGE demonstrated a single band, and the protein used as detecting antigen. Culture engineering bacteria BL21 (PET-32a) and add IPTG to induce protein expression. The protein was used as screen antigen.Fusion protein His-PoIFN-γwas injected to 8-week-old female mice, 100μg pure mouse. And three days after final immunization, spleen cells got from immunized mice were fused with the SP2/0 myeloma cells. With the purified GST-PoIFN-γand unrecombinant protein as detecting antigen, positive hybridoma clones were screened by indirect ELISA. And positive clones were subcloned three times with limiting dilution method. Finally, a hybridoma which stably secrets specific monoclonal antibody against porcine IFN-γwas established,and named B3 strain.The specificity of the McAb was characterized by indirect ELISA, Dot-ELISA and Western-blot. The results demonstrated that the monoclonal antibodies could react specifically to the recombinant His-PoIFN-γand GST-PoIFN-γ, but not irrelative proteins such as His protein, His-ChIFN-γ, GST-ChIFN-γ, His-F and GST-E2, suggesting that McAb was PoIFN-γspecific monoclonal antibodies. The ELISA titers of the McAb ascitic fluids were more than 1:160000. Further characterization revealed that B3 strain belongs to IgG1 isotype, and the light chains areκchain.In conclusion, in present study this gene was successfully expressed and purified, and then we obtained and identified the mouse monoclonal antibody against porcine immunoglobulin, which may have important application value in further studies on immune detection, the functions of immune cells and immune regulation. It is very important for the further research to develop the monoclonal antibody against porcine infectious disease.