Analyse The Sextion Of The Antiviral Activity Of The Bovine IFN-γ By The Phage Display Peptide And Monoclonal Antibody

The purpose of this study is based on the BoIFN-gamma corresponding monoclonal antibodywhich combined with phage peptide to study its antiviral activity section, it provided theory basisfor further research of IFN’s antiviral activity and structure activity relationship.In this study, BoIFN-gamma genes was divided into five sections to express and react with ratanti BoIFN-gamma monoclonal antibody3C2,1H4which saved in the lab, results show that the3C2and1H4specially combined with A1and A3, the binding sites was61-66aa (sequencesKDNQVI) and1-31aa.At the same time, this study established the M13phage ELISA detection method, thepreparation of rabbit anti M13capsid protein antibody, and the antibody purification and tagging,before and after marking purified products, we detected the tagging activity, meanwhile the labeledproduct was determined by western blot and indirect ELISA between the labeled product andpositive phage H11, H12, H14, H16, H18which is saved by lab. The results showed that the rabbitanti-M13serum is produced with titer of more than1:32000and the purification product activitywith titer of1:4000.The identification results between enzyme-labeled antibody and positive phagesaved by lab showed the consistent results, which can explain the enzyme-labeled antibody hascertain application value and used to the BoIFN-gamma combined screening of peptide. WithBoIFN-gamma protein as a target molecule with12random peptide library for screening, afterthree rounds’ selection, production expenses rise gradually, the third round is about8times as thefirst round, illustrates basic objective phage obtained the effective enrichment. Selected20phageclone. And in the appraisal of the ELISA, PCR identification ultimately measured positive phageconsensus sequence for WH*H and HKH. After further competitive inhibition experiment, in whichpositive phage IFN-gamma-3and IFN-15respectively can restrain BoIFN-gamma-combined withprotein A1and A4, specific binding sites is the42-46aa (sequence KKIIQ) and63-66aa (sequenceNQVI) of the BoIFN-gamma amino acid sequence.BoIFN-gamma protein has certain antiviral activity, the antiviral activity in MDBK-VSV is3.1×105U/mg. And at the same time, block experiment was carried out to the BoIFN-gammacorresponding antibody IgG and positive phage IFN-gamma-3and IFN-gamma-15antiviral activity. Blocking effect is3C2>Polyclonal antibody>1H4，IFN-γ-15>IFN-γ-3.After antiviral activityanalysis, specific binding sites is the1-33aa,42-46aa (sequence KKIIQ) and63-66aa (sequenceNQVI) of the BoIFN-gamma amino acid sequence.