关键词: 猪β-干扰素; 毕赤酵母; 分泌表达; 抗病毒活性; 猪繁殖与呼吸综合征病毒

Abstract: Based on the sequences of porcine interferon-β gene and the mature gene (mPoIFNβ) available in GenBank, the gene encoding porcine interferon-β was modified for adapting codons of Pichia pastori and a recombinant vector pPICZαC-PoIFNβ was developed, while maintaining the same protein sequence of porcine interferon-β. The plasmid pPICZαC-PoIFNβ was linearized with SacⅠ and electroporated into Pichia pastoris X-33. The recombinant colonies were selected in high resistance culture of YPDS+Zeocin and identified by PCR. Induced by menthanol, a few positive clones were obtained secretory highly expressed the PoIFNβ protein with a protein concentration of 127.9 mg/L. The expressed supernatant was identified by SDS-PAGE and Western blotting. On the gel and membrane there were two major protein bands with molecular weight about 25 and 28 ku and they showed positive reaction with anti-PoIFNβ antibody. The CPE suppression by PoIFNβ was tested for vesicular stomatitis virus (VSV) in BHK-21 cell line and the porcine reproductive and respiratory syndrome virus (PRRSV) in Marc-145 cell line. The antiviral activity of the expressed PoIFNβ on BHK-21 cell line challenged with VSV was 2.8×10³ IU/mL, and 1.6×10³ IU/mL on the Marc-145 cell line challenged with PRRSV.

Key words: porcine interferon-β; Pichia pastori; secretory expression; antiviral activity; porcine reproductive and respiratory syndrome virus