猪α-干扰素的原核表达及活性测定

目的：表达并纯化出具有生物学活性的重组猪α-干扰素。方法：根据前期合成的猪α-干扰素基因序列设计引物,用PCR方法扩增出猪α-干扰素基因,并将其定向克隆入原核表达载体pET28中,酶切及测序鉴定正确后,转化大肠杆菌BL21进行诱导表达,对表达产物通过镍琼脂糖凝胶柱亲和层析纯化、透析法复性后,采用微量细胞病变抑制法测定重组猪α-干扰素的活性。结果：测序结果表明构建了猪α-干扰素原核表达载体pET28-poIFN-α;诱导表达后经SDS-PAGE分析,在相对分子质量约21×10^3的位置出现明显的诱导蛋白条带,与目的蛋白大小相近;经分析表达产物主要以包涵体形式存在,约占菌体总蛋白的36%,纯化后的目的蛋白纯度约为90%;采用微量细胞病变抑制法测定其活性约为1.67×10^6U/mg。结论：获得了纯度较高并具有较高活性的重组猪α-干扰素,为下一步研究猪α-干扰素药物价值及其生物制剂的生产、应用奠定了基础。

Prokaryotic Expression and Activity Assay of Recombinant Porcine Interferon-Alpha

Objective： To express and purify recombinant porcine interferon-alpha with biological activity. Methods： According to the synthesized interferon sequence, we designed the primers and amplified interferon gene, and then constructed the prokaryotic expression vector pET28-polFN-α. After it was confirmed with restriction nucleases digestion and DNA sequencing, the pET28-poIFN-α was transformed into E.coli BL21（DE3） and induced with IPTG to express the protein of interest. The activity of the purified product was verified by inhibiting the cytopathic effect. Results： Restriction analysis and sequencing proved that the prokaryotic expression vector pET28-poIFN-α was successfully constructed. The expressed product in a form of inclusion body showed a relative molecular mass of about 21 kD, and it was 36% of the total somatic protein. The purified recombinant porcin reached a purity of more than 90% and its activity was up to 1.64×10^6 U/ mg. Conclusion： The recombinant porcine interferon-alpha with high antiviral activity has been got successfully, and it will be developed further.