【摘要】 将AsiaⅠ型口蹄疫病毒YNBS/58株VP1基因和去信号肽的牛α-干扰素基因共同亚克隆于原核表达载体 pET-28a中,转化 BL21 后经 IPTG诱导,实现了重组融合蛋白 VP1-BoIFN -α在大肠埃希氏菌 BL21 中的高效表达,表达产物经 SDS-PAGE 和 Western blotting 分析,重组的VP1-BoIFN-α蛋白分子质量约为 46 ku,与预期大小相符。薄层扫描分析显示,重组蛋白 VP1-BoIFN-α的表达量占菌体总蛋白的30%,且以包涵体的形式存在。包涵体提取物用 8 mol/L尿素溶解后,在变性条件下利用Ni-NTA柱对VP1-BoIFN-α融合蛋白进行了纯化。更多还原

【Abstract】 The VP1 gene of foot-and-mouth disease virus type AsiaⅠ YNBS/58 and the bovine interferon-α (BoIFN-α) gene were amplified from recombinant plasmid pProex-VP1 and pUC-BoIFN-α , respectively, by PCR. After digestion with restriction endonucleases, the VP1 gene and the BoIFN-α gene were subcloned into the prokaryotic expression vector pET-28a, and transformed into BL21(DE3) pLysS (competent) cells. The transformant was induced by IPTG, and the recombinant VP1-BoIFN-α was (expressed) as a fusion protein with a histidine hexamer tag at the N-terminal end of the protein. The fusion protein wasabout 46ku in size and accounted for 30% of the total protein. The recombinant VP1-BoIFN-α protein (aggregated) in Escherichia coli and formed insoluble inclusion bodies. Following solubilization with 8mol/L urea, the inclusion bodies were purified under denaturing conditions by Ni-NTA His Bind Resin and then refolded experimentally.更多还原