Based on the sequences of porcine interferon-p gene and the mature gene (mPoIFNβ) available in GenBank. The gene encoding porcine interferon-p 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 Sac I 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 Lu/Ml on the Marc-145 cell line challenged with PRRSV.%本研究根据GenBank上登录的猪β-干扰素基因成熟肽核苷酸序列(mPoIFNβ),在保持原猪β-干扰素蛋白序列不变的基础上,对猪β-干扰素基因进行了毕赤酵母偏嗜性改造,并构建了毕赤酵母重组表达质粒pPICZαC-PoIFNp.pPICZαC-PoIFNβ经Sac Ⅰ酶切线性化后,电击转化导人感受态的毕赤酵母菌株X-33中,转化子经YPDS+ Zeocin抗性平板筛选和PCR鉴定后获得多株阳性菌株.阳性酵母菌株经甲醇诱导分泌表达了重组PoIFNβ,其表达量约为127.9 mg/L.表达产物经SDS-PAGE和Western blotting检测,结果表明,表达产物为分子质量约25和28 ku的混合物,并且二者都可与PoIFNβ阳性血清结合.以细胞病变抑制法测定重组β-干扰素在BHK-21细胞上的抗水泡性口炎病毒活性为2.8×103IU/mL;对猪繁殖与呼吸综合征病毒(PRRSV)在Marc-145细胞上抗病毒活性达到1.6×103 IU/mL.
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