Prokaryotic Expression of Chicken Interferon-γ and Preparation and Identification of Its Monoclonal Antibodies
[Objective]The purpose of this study was to express chicken interferon-gamma(ChIFN-y)by prokaryotic expression system and prepare its monoclonal antibody to provide crucial reagents for the detection of natural ChIFN-γ.[Method]Recombinant plasmid pET21a-ChIFN-γ was constructed by cloning codon optimized ChIFN-γ gene into pET-21a(+)vector,and then transformed into Escherichia coli BL21(DE3)competent cells.The recombinant protein was induced by IPTG.BALB/c mice were immunized with this purified protein.The splenocytes of immune-qualified mouse was hybridized with myeloma cells(SP2/0)by cell fusion technique.Positive hybridoma secreting specific antibodies against ChIFN-γ were identified through screening using indirect ELISA,and prepare ascites,purified ascites with Protein A/G to obtain specific antibodies.The classes and subclasses of monoclonal antibodies were identified.Western blotting and indirect immunofluorescence assay(IFA)were used to detect the reactivity,affinity,and specific cross reactivity of monoclonal antibodies.Monoclonal antibodies were labeled with horseradish peroxidase(HRP),and antibody pairing were conducted using pairwise methods.[Result]Soluble recombinant ChIFN-γ protein were successfully prepared.Western blotting showed that ChIFN-γ binded to the corresponding antibody,presenting specific bands(16 ku).Seven hybridoma cell lines specific to ChIFN-γ were obtained which were 3E8,5F8,2G12,5 A10,3D8,3B5 and 3G2,respectively.Western blotting and IFA results showed that all 7 monoclonal antibodies had good reactivity,and could specifically recognize eukaryotic expressed ChIFN-γ.The results of class and subclass identification of monoclonal antibodies showed that the heavy chains of 3E8,5F8,3D8,3B5 and 2G12 were all IgG1.The heavy chains of 5A10 and 3G2 were IgG2a and IgM,respectively.The light chains of the 7 monoclonal antibodies were all Kappa type.The 7 monoclonal antibodies obtained had dissociation constants(Kd)between 1.04 and 58.33 nmol/L,indicating high affinity.Five pairs of paired antibodies were also obtained.[Conclusion]This study successfully prepared recombinant ChIFN-γ protein and its monoclonal antibodies,laying the foundation for further research on ChIFN-γ and its related studies in avian immunology,virology and vaccine efficacy evaluation.
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