Abstract

Porcine interferon-α (pIFN-α) could be used as the vaccine adjuvant to enhance the antiviral ability of porcine in swine industry. In here, a combinational strategy integrating codon optimization, multiple gene insertion, strong AOX1 promoter, and efficient secretion signal sequence was developed to obtain high-level secreted pIFN-α in Pichia pastoris GS115. The codon optimized pIFN-α shared 76% sequence identity with the original pIFN-α, which was inserted into the P. pastoris genome under AOX1 d1-2x201 promoter and MF4I secretion sequence. Our results showed positive correlation between the mRNA and secreted protein levels with the copy numbers of genome-integrated pIFN-α gene in the recombinant P. pastoris strains. The recombinant opt-pIFN-α-6C strain bearing six copies of pIFN-α expression cassette produced the highest extracellular secretion of pIFN-α of 3.2 ± 0.1 mg/mL in shake flask experiment, and 17.0 ± 0.8 mg/mL in a 5 L high-cell-density cultivation after methanol induction of 84 h. The antiviral activity of secreted pIFN-α from the high-cell-density cultivation was determined to be approximately 2.8 ± 0.9 × 10 9 IU/mL against the vesicular stomatitis virus (VSV) infected Madin-Darby bovine kidney (MDBK) cells. This strategy provided an efficient way to generate recombinant P. pastoris strains in a non-antibiotics-selection manner, which might also give general guidance for the heterologous expression of other proteins in P. pastoris .