猪α干扰素的原核表达及抗高致病性猪繁殖与呼吸综合征病毒活性测定

Antiviral activity of the porcine interferon-alpha protein against high-pathogenic porcine reproductive and respiratory syndrome virus

为了研究高效广谱的基因工程抗病毒制剂,本研究采用PCR技术自猪肝脏总DNA中扩增、克隆猪α干扰素(PoIFN-α)成熟肽基因并亚克隆入pQE30载体进行原核表达,对表达的融合重组猪α干扰素(rPoIFN-α)蛋白通过尿素变性、低浓度蛋白复性液复性、PBS溶液透析等步骤进行纯化,采用细胞病变抑制法分别测定rPoIFN-α蛋白在PK15细胞上抗水泡性口炎病毒(VSV)增殖活性及在Marc-145细胞上抑制高致病性猪繁殖与呼吸综合征病毒(PRRSV)的增殖活性,以分别评价rPoIFN-α的活性单位及其体外抑制高致病性PRRSV增殖所需的最低浓度。结果表明,克隆的PoIFN-α成熟肽基因全长501bp,编码166个氨基酸;表达的rPoIFN-α蛋白分子量大小20.7ku左右,与预期大小相一致;经纯化后的蛋白纯度在95%以上;纯化的rPoIFN-α蛋白在PK15细胞上抗VSV病毒活性单位为1.4482×104IU/mL(5.2×106IU/mg),在Marc-145细胞上能完全抑制高致病性PRRSV增殖所需的rPoIFN-α蛋白最低有效剂量为640U/mL(2.3×104U/mg)。这些研究结果为新型基因工程抗病毒制剂的开发以及用于高致病性PRRSV性疾病的预防和治疗奠定了基础。

To exploit a genetic engineered antiviral medicine against the porcine immune suppression diseases, mature porcine interferon-alpha （PolFN-α） gene was amplified by PCR. The PCR product, which comprised 501 bp nucleotides encoding 166 amino acids, was sequenced and cloned into the prokaryotic expression vector pQE30. The expressed recombinant PolFN-α（rPolFN-α ） protein was pttdfied under denatured condition and refolded in a novel re-naturation buffer. The CPE suppression by rPolFN-α was tested for vesicular stomatitis virus （VSV） in porcine kidney 15 （PK15） cell line and the high-pathogenic porcine reproductive and respiratory syndrome virus （PRRSV） in Marc-145 cell line. The antiviral activity of rPolFN-α on PK15 cell line was 1.4482 × 10^4 IU/mL, and the lowest effective dose of rPolFN-α to inhibit the proliferation of high-pathogenic PRRSV was 640 U/mL （2.3 × 10^4 U/mg）. These results indicated that interferon-alpha could be used as an antiviral agent to interfere the high-pathogenic PRRSV and other porcine virus infections.