High-level Secretory Expression Of Bavine Interferon α In Pichia Pastoris And Its Evaluation Of Bioactivity

China is a big country in cattle industry, but some infectious diseases limit the development of cattleindustry, such as the cow mastitis, foot and mouth disease, bovine viral diarrhea. Some researches showthat recombinant bovine alpha interferon (boIFN-) plays an important role in antiviral activity againstthe vesicular stomatitis virus, foot and mouth disease virus, bovine viral diarrhea virus and so on in vitro.High-level secretory expression of interferon will benefit the control and prevention of some infectiousdiseases. Pichia Pastoris (P.pastoris) expression system as a kind of mature eukaryotic expression toolhas been used to successfully secretary expressed a variety of foreign proteins. In this research boIFN-was efficiently secretory expressed in P.pastoris which laid a solid foundation for the production andlarge-scale application of boIFN-, provided the theoretical basis and experimental basis for bovineinterferon.In the research, firstly to achieve high-level secretory expression of boIFN-in P.pastoris, theboIFN-gene was optimized through four aspects including the codon bias optimization, adjustment ofG+C content, the adjustment of AT rich region, then the optimized bovine interferon alpha wasartificially synthesized and inserted into the secreted expression vector pPIC9K. Recombinant plasmidwas transformed into P.pastoris GS115by electroporation and the positive recombinant strainsP.pastoris were screened with0.25mg/mL to4mg/mL of G418and MD plates which were capable ofexpressing boIFN-in culture medium detected by SDS-PAGE and western blot. In addition, boIFN-secretory expressed by recombinant P.pastoris was purified by Ni+column, fermentation conditionswere optimized through five aspects: culture time, speed, temperature, inducing methanal concentration,transferring concentration (OD/mL). The concentration of purified protein was detected by A280assay.Furthermore, antival activity was tested, compare the antiviral activity between boIFN-and poIFN-inMDBK and IBRS-2cell line.Results show that the nucleic acid homology between the optimized genes and original sequencewas73.1%, protein sequence remains the same.134bp were optimized affecting the encoding of110amino acids, the adjusted the G+C content is39.7%. Optimized boIFN-was successfully cloned intovector pPIC9K. Recombinant plasmid pPIC9K-opti-boIFN-was successfully reconstructed. Positivestrains were screened by SDS-PAGE and western blot, boIFN-was succesfully secretory expressed byP.pastoris. It is found that19kD protein bands was detected which corresponds to boIFN-protein. Inaddition, approximate200μg/mL of boIFN-was secretory expression from recombinant P.pastorisunder the optimized conditions of the fermentation by incubating at26℃for72hours with250r/min,inducing with1%of methanol, the last transferring concentration1.0OD/mL(2%transferringpropotion). Furthermore, the boIFN-was able to efficiently inhibiting virus replication in recombinantvesicular stomatitis virus(VSV-GFP)/MDBK cells detection system. The antiviral unit of originalboIFN-and purified boIFN-is2×106AU/mL and107AU/mg separately in MDBK cells. Theantiviral unit of original boIFN-and purified boIFN-is2×105AU/mL and105AU/mg separately inIBRS-2cells. In this research boIFN-alpha was efficiently secretory expressed in P.pastoris which laid a solid foundation for the large-scale production and provided the theoretical basis for clinicalapplication.