The Study On Expression Of Porcine Interferon Alpha/Beta/Gamma Gene In 293T Cell

IFN(Interferon,IFN) is a kind of anti-virus,regulating cell growth and differentiation,immune-modulated protein.In veterinary medicine,it is important to prevent disease and cure virus-disease,to study vaccine adjuvant et al.Relatively speaking,the studies on the animal IFN begin comparatively late in veterinary medical circles.In pork-producing community,various infectious diseases which are caused by viral or bacterial pathogens,badly restricted the developmengt of natural and regional farming industries,and also seriously affect human health in whole world.hence,it's necessary and great meaningful in both economic development and societal stabilization to prevent and control infectious diseases.In light of the consideration,in this study porcine interferon-alpha(PoIFN-α),porcine interferon-beta (PoIFN-β) and porcine interferon-gamma(PoIFN-γ) were investigated.The PoIFNs were respectively subcloned into pcDNA3.1-EGFP and rAAV and constructed pcDNA3.1-EGFP-PoIFN-α/β/γ(named pcDNA3.1-IFN-α/β/γ)和rAAV-PoIFN-α/β/γ(named AAV-PoIFN-α/β/γ) then six eukaryotic expression plasmids respectively transfected into 293T cell,The culture supernatants were detected with SDS-PAGE.The main researches included as below:1.Three pairs primmer were respectively designed in according to PoIFN total length which was amplified by our lab and NheⅠand HindⅢwere designed in the primmers.PoIFN-α/β/γsegments of sequences was amplified by PCR,three segments of sequences were subloned to pcDNA3.1-IFN vector,and those linked vectors were transformed into E.coli DH5α.The positive clones were verified from PCR with bacterium,the conclusions of sequence determined showed that poIFNs were cloned. Bacterium was amplified culture then extract plasmid and purfied the plasmids.2.Three pairs primmer were respectively designed in according to PoIFN total length which was amplified by our lab and EcoRⅠand HindⅢwere designed in the primmers.PoIFN-α/β/γsegments of sequences was amplified by PCR,three segments of sequences were subloned to AAV vetor,and those linked vectors were transformed into E.coli.DH5α.The positive clones were verified from PCR with bacterium,Amplifying culture then extract plasmid and purfied the plasmids.3.Transfection of PoIFN in 293T cellThree plasmids system(AAV-PoIFN,AAV-RC,AAV-pHelper,mixed with 1:1:1) and pcDNA3.1-PoIFN plasmids were transfected in 293T cell wirh lipofectamine^TM2000.The IFN protein was detected by western-blot.