Secreted expression of canine interferon alpha subtype 1 in Pichia pastoris and its antiviral activity

Canine interferon alpha subtype 1(CaIFN-α1)mature protein encoded gene was sub-cloned from pMD18-T-CaIFN-α which contain complete gene of CaIFN-α1.The sub-cloned gene was inserted into pPICZα-A vector and resulted in pPICZα-A-CaIFN-α1.The recombinant plasmid was linearized with Sac Ⅰ and was then transformed into Pichia pastoris X-33 strain by electroporation.The existing of CaIFN-α1 gene in the transformed yeast cells was detected by PCR using CaIFN-α1 primers.Whether the CaIFN-α1 gene was integrated into the alcohol oxidase promotor(AOX1)locus on yeast chromosome was verified by amplification with AOX1 primers.The identified transformants were isolated and assayed for the expression of CaIFN-α1.The expression of CaIFN-α1 was induced with methanol.The existing of CaIFN-α1 in the supernatant of culture was firstly detected through SDS-PAGE,and then the positive samples were further identified by antiviral activity testing.There was a protein with relative molecular weight of approximately 2.7×104 in the supernatant.In contrast with the deduced theoretical molecular weight of CaIFN-α1,the expressed protein was larger which may be due to the difference of glycosylation.The content of the secreted CaIFN-α1 was about 96 mg·L-1 and its antiviral activity was about 1.45×106 U·mL-1.The specific activity of porcine interferon beta produced by the Pichia pastoris was approximately 1.49×107 U·mg-1.Bioactivity of CaIFN-α1 was highly species-specific,which showed high antiviral activity in canine cell MDCK and low antiviral activity in CEF(chicken),but not in the F81(feline)and MDBK(bovine).The antiviral activity of recombinant CaIFN-α1 aganist canine distemper virus(CDV)and pseudorabies virus(PrV)was also assessed in the present experiment.And the result indicated that CaIFN-α1 could effectively inhibit the replication of CDV and PrV in MDCK cells.