关键词: 猪α干扰素, 毕赤酵母, 分泌表达

Abstract: Porcine interferon-alpha（PoIFNα）was expressed and purified to evaluate its functions. The PoIFNα gene，amplified by PCR，was cloned into P. pasteris expression vector pPIC9k which had been digested by SnaBⅠ and EcoRⅠ enzymes. The transfer plasmid pPIC9k/PoIFNα was verified by enzyme digestion and sequencing. The pPIC9k/PoIFNα linearized by SalⅠ was electro-porated into P. paseris strain GS115. The recombinant yeast GS115/PoIFNα with four-copied PoIIFNα genes was isolated on the YPD agar plate with 1 mg·mL^-1 G418. The recombinant protein PoIFNα was expressed in the fermentation supernatant at a high level of 136 mg·L^-1 after being induced by methanol. rPoIFNα was isolated at a purity of 81% by one step of SP-Sepharose chromatography. As a result，the purified product was verified to be of high cytokine activity by inhibiting the VSV viral cyto-pathogenic effect on MDBK cell culture，which was about 2.27×108 U·mg^-1.

Key words: porcine interferon-alpha, Pichia pasteris, secreted expression