Modification and Expression of Canine Interferon α1 Gene in Pichia pastoris

Objective To modify canine interferon α1(CaIFNα1) gene so as to increase its expression level in Pichia pastoris and its antiviral activity.Methods According to the codon bias of P.pastoris,CaIFNα1 gene was modified and synthesized and cloned into secretory expression vector pPICZαC.The constructed recombinant plasmid pPICZαC-CaIFNα1was transformed to P.pastoris X33 by electrotransformation for expression under induction of methanol.The expressed product was identified by SDS-PAGE and subjected to amino acids sequencing,then determined for expression level and activity against vesicular stomatitis virus(VSV).Results Both restriction analysis and sequencing proved that recombinant plasmid pPICZαC-CaIFNα1 was constructed correctly.The amino acid sequence of expressed recombinant CaIFNα1,with a relative molecular mass of about 24 000,was identical to that of natural CaIFNα1.The protein contents of three batches of expressed products in P.pastoris were 0.253,0.198 and 0.224 mg / ml respectively.All the three batches of expressed protein showed no anti-VSV activities in Vero cells,of which the activities in MDCK cells were 1.58 × 108,1.30 × 108 and 1.50 × 108 U / mg respectively.Conclusion CaIFNα1 gene was successfully modified and highly expressed in P.pasoris,which laid a foundation of large-scale production of interferon by fermentation.