关键词: 西伯利亚鲟, IFN-γ基因, 原核表达, 重组蛋白, 单克隆抗体

Abstract: Using transcriptome information, the readable coding frame (ORF) 528 bp encoding 176 amino acids ofIFN-γofAcipenser baeriiwas cloned with the characteristic sequence and nuclear localization sequence of IFN-γ. According to this sequence, a prokaryotic expression vector Pet30α-IFN-γwas constructed containing 6His (histidine) at the n-terminal. Then the Pet30α-IFN-γplasmid was transformed intoEscherichia coliBL21(DE3), which was induced by IPTG (isopropyl-β-d-thiogalactoside), and the expressed recombinant 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 ofAcipenser baerii.

Key words: Acipenser baerii;IFN-γ, prokaryotic expression, recombinant protein, monclone antibody