【摘要】 将水牛γ-干扰素成熟蛋白(IFN-γ)基因亚克隆至pGEX-6P-1中,成功构建了pGEX-BoIFN-γ表达载体,转化至大肠杆菌BL21(DE3)重组菌进行IPTG诱导表达。应用SDS-PAGE方法对表达产物rGST-BoIFN-γ进行分析,用GST琼脂糖凝胶柱对可溶性蛋白进行分离纯化,并采用ELISA和Western-blot试验鉴定重组蛋白的免疫活性。将可溶性纯化蛋白作为抗原免疫小鼠,包涵体蛋白经SDS-PAGE分析后切取目的蛋白免疫小鼠以制备多克隆抗体。结果表明:重组菌经37℃0.5 mmol/L的IPTG诱导3 h,表达出43 ku的融合蛋白。目的蛋白的表达量达到菌体总蛋白量的30%,分别以可溶性和包涵体两种形式存在,而可溶性表达蛋白纯化后的纯度较高。获得的重组蛋白具有良好的免疫活性。均能从可溶性蛋白及包涵体蛋白免疫小鼠得到针对rGST-BoIFN-γ的特异性多克隆抗体。更多还原

【Abstract】 The water buffalo mature peptide IFN-γ gene was subcloned into the expression plasmid pGEX-6P-1,and then the constructed pGEX-BoIFN-γ was transformed into Escherichia coli BL21(DE3).The GST-fusion protein was expressed highly under IPTG induction.The soluble fusion protein was purified using GST affinity columns.SDS-PAGE,Western-blot and ELISA tests were employed to determine the expression,purification and activity of the expected protein,respectively.BALB/c mice were immunized with the purified soluble portion protein and the inclusion bodies protein in gel slices to generate anti-mouse rGST-BoIFN-γ polyclonal antibody.The results showed that the target protein expressed at 37 ℃ for 3 h under induction with 0.5 mmol/L IPTG in E.coli BL21(DE3) was 43 ku in molecular mass,made up 30% of the whole bacterial proteins,and existed in soluble portion and inclusion bodies.The ELISA and Western-blot indicated that the recombinant protein possessed good immunological activity.更多还原