鸡朊蛋白的原核表达及其抗血清的制备  

Prokaryotic expression of chicken prion protein and preparation of its antiserum

作　　者：罗鹏[1] 吴润[1,2] 刁小龙[1] 管宏伟[1] 张莉[1] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2012年第3期268-274,共7页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31160510);甘肃省自然科学研究基金计划项目(1107RJZA198);家畜疫病病原生物学国家重点实验室开放基金项目(SKLVEB2009KFKT012)

摘　　要：运用分子生物学方法分别构建了pGEX-4T-ChPrP1、pGEX-4T-ChPrP2、pGEX-4T-ChPrP3重组表达载体,经转化大肠杆菌BL21(DE3)和IPTG诱导表达,并对融合蛋白诱导表达时间、IPTG浓度进行优化,诱导表达的GST-ChPrP(25～247)融合蛋白经GST-trap FF柱亲和层析纯化,用纯化后的GST-ChPrP(25～247)融合蛋白免疫新西兰雄兔,以探究鸡朊蛋白的结构与功能。结果表明,本试验成功构建了pGEX-4T-ChPrP1、pGEX-4T-ChPrP2、pGEX-4T-ChPrP3重组表达菌,SDS-PAGE分析结果显示,得到大小为50、42和32ku左右的融合蛋白GST-ChPrP(25～247)、GST-ChPrP(96～247)、GST-ChPrP(174～247),与预期结果基本相符,在最适条件下每升pGEX-4T-ChPrP1菌液通过GST-trap FF层析柱获得的GST-ChPrP(25～247)融合蛋白达10mg,制备的抗血清经琼脂糖凝胶扩散试验检测到较高的多克隆抗体效价(1∶32);Western-blot分析结果显示,融合蛋白GST-ChPrP(25～247)、GST-ChPrP(96～247)和GST-ChPrP(174～247)及鸡脑组织蛋白均可与该抗血清发生特异性免疫反应。证实,鸡重组朊蛋白具有良好的抗原性,与鸡脑组织朊蛋白具有相同的抗原成分。To study the structure and function of chicken prion protein（ChPrP）,three recombinant expression vectors（pGEX-4T-ChPrP1,pGEX-4T-ChPrP2 and pGEX-4T-ChPrP3） were constructed,and were transformed into Escherichia coli BL21（DE3）,respectively.The fermentation conditions including time and IPTG concentration were optimized.Fusion protein GST-ChPrP（25-247） of pGEX-ChPrP1 was purified by GST-trap FF affinity chromatography and was used to immunize New Zealand male rabbits.The results showed that an efficient prokaryotic expression system of pGEX-4T-ChPrP1,pGEX-4T-ChPrP2,and pGEX-4T-ChPrP3 was successfully constructed.SDS-PAGE analysis showed that three IPTG-induced fusion proteins GST-ChPrP（25-247）,GST-ChPrP（96-247）,and GST-ChPrP（174-247） were expressed as expected with the molecular weight of about 50,42,and 32 ku,respectively.Under optimal fermentation conditions,10 mg fusion protein GST-ChPrP（25-247） could obtain by GST-trap FF affinity chromatography in one liter bacilli of pGEX-ChPrP1.The results of agar diffusion test showed that prepared polyclonal antiserum has a high antibody titer（1∶32）.Western-blot analysis proved that the three expressed fusion protein and the extration of chicken brain tissue can specifically reacted with the antiserum respectively, demonstrating that the recombinant chicken prion protein possesses good antigenicity and the same antigenic components in brain tissue.

关 键 词：鸡 朊蛋白 原核表达 抗血清 抗原性 

分 类 号：S859.797[农业科学—临床兽医学]