Research On Utilization Of Phage Display Technology To Screening Of Human IFN-? Epitope

Interferon(IFNs)is a kind of cytokines that secreted by immune cells,and there are currently 3 types of interferon known.IFN-?is the only member of the class II interferon family and is also called immunointerferon because of its outstanding immunomodulatory ability.Although IFN-?plays an important role in innate and acquired immune responses,tumor surveillance,as well as combating viral and bacterial infections,overexpression of IFN-?can also cause many diseases.The use of anti-IFN-?monoclonal antibodies(mAbs)to neutralize excessive IFN-?in the body is considered to be an effective treatment.However,when murine mAbs or chimeric antibodies are used in humans,they will cause different degrees of immune response in the body,which not only affects the efficacy of the drug,but also causes allergic reactions.Therefore,fully human mAb drugs with low immunogenicity have been paid more and more attention by researchers and medical workers.Using transgenic mice to make fully human mAbs is a mature and reliable method,but the widespread use of this method has been limited due to its long research and development cycle and huge capital investment.Therefore,many researchers have focused on phage display technology,which is an in vitro screening technology that selects the target polypeptide or protein from a large number of variant colonies.The human immunoglobulin sequence can be used to construct a fully human antibody library,and then use the peptide or protein as a target to screen out fully human mAbs.In this study,the full-length fragment of human IFN-?gene was amplified using the laboratory's own plasmid pET28a-IFN-?as a template,and a 50-200 bp gene fragment was randomly digested by DNase I.The gene fragment was connected to pComb-EcoRV vector,and then transferred into TG1 competent cell by electrotransformate to construct a phage gene fragment peptide library.The library capacity is 5.1×10~6 cfu/mL,and the monoclonal genes are randomly selected for sequencing,and the library accuracy rate is 10%.Then,using the anti-IFN-?murine mAb obtained in the laboratory as a target,the phage peptide library was subjected to five rounds of biological panning,and the enrichment effect was detected by polyclonal phage ELISA to determine the panning effect.Then,using the monoclonal phage ELISA results as a reference,select high optical density value monoclonal for gene sequencing.Finally,four clones with homologous sequence results as IFN-?were obtained,suggesting that the four sequences are potential epitopes corresponding to the anti-IFN-?mAb.In summary,this study laid the foundation for the follow-up research on the screening of fully human anti-IFN-?mAbs using phage antibody library.