Expression,purification and antiviral activity analysis of the chicken interferon alpha mature protein expressed in pichia pastoris

In order to construct recombinant yeast strains expressing extracellular ChIFNα,the recombinant expression vector pPIC-ChIFNα of chicken interferon alpha mature protein gene was transformed into Pichia pastoris GS115 strain by electroporation to integrate with the genome,many positive Pichia pastoris strains were obtained by PCR analysis.The transformants with multicopy were screened by high concentration G418 and induced to express ChIFNα with methanol.Two integrants with high expression level were obtained,named GS-ChIFNαB_1 and GS-ChIFNαB_2.The culture supernatant of high expression strains was concentrated by PEG20000,purified with DEAE Sepharose Fast Flow and Sephadex G200,and the antiviral activity of secretion supernatant was detected by vesicular stomatitis cytopathic effect inhibition assay(CPE).The assay results revealed that the recombinant ChIFNα gene was expressed highly and secreted effectively in Pichia pastoris,and the expressed product had normal bioactivity.The antiviral activity units was 570×10~4 U/ml,and the specific activity of recombinant ChIFNα was up to 2 980×10~4 U/mg protein.