Expression of Huiyang chicken IFNγ gene in Pichia pastoris and analysis of antiviral activity of the recombinant protein

To identify antiviral activity and the secondary structure of the recombinant protein of Huiyang chicken IFN-γ (chIFN-γ2) expressed in Pichia pastoris,the cDNA encoding mature peptide of IFN-γ was subcloned from plasmid pGEM-T-chIFN-γ2 and recombined with vector pPICZαB.The recombinant plasmid confirmed by enzyme digestion,PCR amplification and sequencing was transformed into P.pastoris GS115.After methanol induction,SDS-PAGE analysis of the culture supernatant of recombinant yeast strains indicated that the yield of recombinant IFN-γ was 0.604mg/mL and the molecular weight was(approximately) 33ku.The recombinant IFN-γ expressed in P.pastoris had antiviral activity of 3.2×10~4(U/mg) in CEF-VSV.The secondary structure of the recombinant IFN-γ was investigated by Circular Dichroism,and contents of α helix,β sheet,turn,and random were 55.1%,8.3%,13.9% and 22.8%,respectively.These results showed that the recombinant IFN-γ with high antiviral activity was expressed successfully in P.pastoris and the chicken IFN-γ was a helical protein which was similar to IFN-γ of mammals.