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

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.