Cloning,prokaryotic expression and monclone antibody prepartion of IFN-γ of Acipenser baerii
Using transcriptome information,the readable coding frame(ORF)528 bp encoding 176 amino acids of IFN-γ of Acipenser baerii was cloned with the characteristic sequence and nuclear localization sequence of IFN-γ.According to this sequence,a prokaryotic expression vector Pet30α-IFN-γ was constructed containing 6His(histidine)at the n-terminal.Then the Pet30α-IFN-γ plasmid was transformed into Escherichia coli BL21(DE3),which was induced by IPTG(isopropyl-β-d-thiogalactoside),and the expressed recombi-nant protein was identified by SDS-PAGE electrophoresis and Western Blot.The recombinant protein was 22.8 ku,mainly existing in the form of inclusion bodies,and the optimal expression condition was induced at 37℃with 0.75 mmol/L IPTG for 6 h.The purified recombinant protein was obtained by nickel column chromatography.The recombinant protein was immunized to mice,8 positive cell lines were obtained by cell fusion,monoclonal antibody purified by protein G affinity chromatography,the valence of antibody was 2×105,which laid a foundation for further study of the immunological function of IFN-γ of Acipenser baerii.
万方数据
维普
