Abstract:

Chinese cow interferon-gamma (ccIFN-γ) gene was amplified by reverse transcription polymerase chain reaction (RT-PCR) from total RNA extracted from lymphocytes in the peripheral blood of bovine. The products of RT-PCR were cloned into pMD18-T vector and then sequenced. The sequencing result showed that the ORF of ccIFN-γ gene was 501bp, encoding 166 amion acids. The initiative 23 amino acids consist of signal peptide, the following 147 amino acids consisting of mature peptide. There are 2 N-glycosylation sites. The second and third molecule structure showed that ccIFN-γ has 7 α-helix that gathered and formed the structure of midheaven. Then mature peptide encoding sequence was inserted into the expressing plasmid pET-28a for expression in E.coli. SDS-PAGE results showed that the recombinant protein was successfully expressed after IPTG induction, with a molecular weight of 20.6 kDa as expected. Western blot analysis showed that the recombinant protein of ccIFN-γ is bovine IFN-γ. The experiment lays a foundation for the further study of bioactivity of recombinant bovine IFN-γ.