Expression Of Porcine Interferon-α Gene In Pichia Pastoris And Determining Of Anti-PRV Activity

China's porcine virus diseases become more and more complex.Porcine interferon-α(PoIFN-α)is a broad spectrum of cytokine that plays important roles on anti-virus,anti-tumor and immune regulation agent.In order to develop the application of recombinant PoIFN-αpreparations,to prevent porcine viral infectious disease,and to further expound the research of cytokines in the field of molecular biology,mature PoIFN-αwas cloned and expressed in pPICZαC expression system in the study.A pair of primers containing EcoRI and KpnI restriction sites were designed,according to PoIFN-αgene in GenBank.Then PoIFN-αgene was cloned from porcine gene DNA by PCR.The PCR product was inserted into pMD18-Simple T Vector,and sequenced.PoIFN-αwas obtained. After the target gene and pPICZαC were digested with EcoR I and KpnI,two DNA fragments were linked.Positive clones were screened in low salt LB with 25 ug/mL zeocin^TM,and analysed by PCR and digestion of restriction enzymes.Positive clones were sequenced.The result showed that IFN-αgene was correctly inserted into the pPICZαC vector.The plasmid of pPICZαC-IFNαwas linearized by Pmeâ… and then transformed into Pichia Pastoris X33 by electroporation after purification.A lot of colonies grew up on the YPDS+Zeocin^TM(100μg/mL)selective culture medium.Some colonies with highly fast multiplying in BMGY,then were expressed some protein in BMMY.After four days induction by 1.0%methanol,the expressed protein of PoIFN-αgene was conformed by SDS-PAGE and Western-Blot.The antiviral activity was assayed by inhibition of the cyto-pathogenic effect(CPE)of Pseudorabies Virus on BHK-21 cells.The results showed that the mature protein gene of PoIFN-αincluding stop coden was about 501bp,shared 97.2%～99.2%homology with the sequence of PoIFN-αgene published in GenBank and the predicted mature protein was 166 amino acids and a stop codon in length.The supernatant was analyzed by SDS-PAGE and Western-Blot.The results showed that the relative molecular weight of the expression protein was about 19.4KDa.The rPoIFN-αprotected BHK-21 cells against Pseudorabies Virus(PRV)early infection in vitro.The highest antiviral titre of rPoIFN-αwas 1.817x10⁶IU/ml.In conclusion,we had expressed porcine IFN-αsuccessfully in Pichia pastoris and the protein has potential application influence on porcine disease prevention and treatment.The experiment lays a foundation for lots of production of the interferon-alpha.