The Expression Of Channel Catfish Interferon Alpha(IFN-α)Gene In Pichia Pastoris

For prevent viral diseases and serve for channel catfish's cultivation,the study focused on channel catfish alpha IFN and hopes to express it in Pichia pastoris expression system.A pair of primers P1/P2 was designed according to the reported nucleic acid sequence of catfish alpha IFN. After analyzing the multitude cloning sites of pPICZαA vector, restriction enzyme Xho I was introduced to upper primer P1 and Not I to the downstream primer P2. The channel catfish alpha IFN gene was amplified from the plasmid pET-IFN-αwith the above primers, the length of open reading framework(ORF)is 492bp. After the target gene and pPICZαA were double digested with Xho I and Not I , the two DNA fragments were linked together. Then the recombinant expression plasmid pPICZα-IFN-αwas sequenced by Invitrogen corporation. The result showed that the target gene was correctly inserted into the pPICZαA vector.The recombinant expression plasmid of pPICZα-IFN-αwas linearized by SacI and then transfomred into Pichia pastoris X33 by electroporation after purification. Colonies grew on the Zeocin selective culture medium with high resistance were chosen by increasing the concentration of Zeocin and induced by 1% methanol in BMMY culture medium. The supernatant which was centrifuged from the culture solution was analyzed by SDS-PAGE. The result showed that channel catfish alpha IFN had been expressed successfully in Pichia pastoris and the protein for the prevention and treatment of viral diseases has potential clinical value.