水牛IFN-γ成熟蛋白基因的原核表达及其产物多克隆抗体的制备  

Expression of interferon-gamma mature protein gene of water buffalo in Escherichia coli and preparation of its polyclonal antibody

作　　者：徐贤坤[1,2] 熊毅[2] 龙剑明[3] 刘棋[2] 曾宪垠[1] 

机构地区：[1]四川农业大学生命科学与理学院,四川雅安625014 [2]广西动物疫病预防控制中心,广西南宁530001 [3]广西大学动物科学技术学院,广西南宁530005

出　　处：《中国兽医科学》2008年第11期1003-1008,共6页Chinese Veterinary Science

基　　金：广西科技厅科技攻关项目(桂科攻0719004-3F)

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。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 antimouse rGST-BolFN-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.

关 键 词：γ-干扰素成熟蛋白基因 大肠杆菌表达系统 水牛 多克隆抗体 BALB/C小鼠 

分 类 号：S823.83[农业科学—畜牧学]