| Interferon(IFN)was initially found to have the function of interfering or preventing the replication of virus.Interferon-γ(IFN-γ)is the only member of the type II interferon family,and its molecules present with three sizes under the normal circumstances due to different degrees of glycosylation.In the case of innate immunity,the secretion of IFN-γis mainly regulated by natural killer cell and natural killer T cells.And in adaptive immunity,IFN-γis mainly produced by CD8~+and CD4~+T cells through paracrine pathways.At different concentrations of IFN-γ,different signaling pathways are activated:the JAK/STAT pathway is activated at high concentrations of IFN-γwhile the activation of ICAM-PI3K-Akt Notch 1 signal will be induced at a low concentration of it.IFN-γcan act on the cells which secrete itself to form the positive feedback,and it can also act on immune cells,helping to identify and eliminate pathogens.As a cytokine that regulates human physiological activities,IFN-γhas a dual impact on the organism and plays an important role in infectious diseases,graft versus host diseases,autoimmune diseases,tumor immunity.Phage display technology combines polypeptides,proteins or antibody fragments with one of the phage shell proteins and expresses them on the surface of the phage shell protein.Compared with the traditional antibody preparation technology,phage display technology can not only screen more kinds of antibodies,but also has the advantages of short screening cycle,low cost,wide application range,and easy humanization of the screened antibodiesBased on phage display technology,this study prepared the blocking antibodies targeted human IFN-γand established the detection method of IFN-γ.After purifying IFN-γthrough eukaryotic expression,we immunized animals with this protein and screened 11 different single-chain antibody sequences targeted IFN-γby phage display technology.Then,we expressed and purified them through the secretory expression and intracellular soluble expression of Escherichia coli.With these 11 antibody sequences,we screened for antibody pairs target IFN-γwith the highest affinity and meeting experimental requirements,in order to develop more economical and accurate detection systems in the future;Meanwhile,through flow cytometry analysis,we analyzed the blocking ability of different antibodies for the binding of IFN-γto its receptor at the cellular level.The main results are as follows:1)Purification of human IFN-γprotein with His and h Fc tags using HEK 293F cells.2)After immunizing chicken with 2 kinds of IFN-γprotein,we established and screened the phage library of chicken antibodies,and 11 chicken derived antibody sequences(C03,C04,C06,C08,C11,C12,C13,C15,C25,C35,C48)which were representative and targeted IFN-γwere obtained.3)22 antibody sequences,which purity and activity met the expectations,with two different tags were purified by secreting expression of Escherichia coli.4)The affinity of each antibody sequence was detected by Elisa and Western Blot.The results showed that all 11 antibody sequences were positive for IFN-γand 5sequences of them can bind to linear epitope.5)Four groups of antibody pairs with high affinity were screened by the sandwich Elisa method and the highest binding antibody pair was selected by the software analysis for subsequent development.6)The cell line with the highest expression of IFN-γreceptors was selected from 5cell lines through flow cytometry analysis.7)2 antibody sequences with the best blocking effect were selected by flow cytometry analysis,as well as five other sequences with blocking effect. |
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