甲型鸭肝炎病毒2A蛋白的原核表达、纯化及多克隆抗体的制备  

Expression,purification and polyclonal antibody preparation of Duck hepatitis type A virus 2A protein

作　　者：胡文[1,2] 孟春春[2] 李传峰[2] 陈宗艳[2] 梁瑞英[2] 李宁[2] 黄云秀[2] 吴润[1] 刘光清[2] 

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《甘肃农业大学学报》2014年第6期5-9,共5页Journal of Gansu Agricultural University

基　　金：国家自然科学基金(31270194;31101848;31300141);农业公益性行业科研专项课题(201003012;201303046);国家高科技研究发展计划863计划(2011AA10A209)

摘　　要：采用RT-PCR方法从甲型鸭肝炎病毒ZJ-V株的RNA模板中扩增出2A基因,将其克隆到表达载体pET-30(+)中,经酶切和测序鉴定后转化表达宿主菌BL21(DE3)细胞中,以IPTG诱导表达His-DHAV-2A融合蛋白.结果表明:目的蛋白在1mmol/L IPTG诱导4h的情况下以可溶性形式表达.表达产物经Ni柱纯化后获得了高纯度的融合蛋白.以纯化后的His-DHAV-2A融合蛋白为抗原免疫白兔制备多抗,Western-blot试验表明制备的多抗可与目的蛋白发生特异性反应.以上结果说明,DHAV的2A蛋白在大肠杆菌中成功表达,且制备的多抗血清可用于2A蛋白的表达检测.Using the total RNA of Duck hepatitis A virus （DHAV）ZJV strain as template,2A gene was amplified by RT-PCR and sub-cloned into pET30a to construct a recombinant prokaryotic expression plasmid pET30a-2A.After sequencing,the recombinant plasmid was transformed into E.coli BL21（DE3）. The transformed bacteria were induced by 1 mmol/L IPTG with 4 hour then the fusion protein was ex-pressed as a soluble.Recombinant protein was purified by Ni-NTA-Resin,and then vaccinated rabbits three times for preparation polyclonal antibody.The results of Western-blot assay indicated the prepared poly-clonal antibody reacted with target protein specificity.These results showed that 2A protein was success-fully expressed in E.coli,and the antiserum could be used for establishment methods for DHAV-2A pro-tein detection.

关 键 词：甲型鸭肝炎病毒 2A蛋白 原核表达 蛋白纯化 多克隆抗体 

分 类 号：S851.3[农业科学—预防兽医学]