鸭源呼肠孤病毒Sigma A蛋白的原核表达及免疫活性检测  

Prokaryotic expression of Sigma A protein of duck reovirus

作　　者：朱英奇[1,2] 陈宗艳[2] 李传峰[2] 孟春春[2] 王桂军[1] 刘光清[2] 

机构地区：[1]安徽农业大学动物科技学院,安徽合肥230036 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国预防兽医学报》2013年第10期849-851,共3页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31270194;31101848);公益性农业科研专项(201003012);国家863计划(2011AA10A200)

摘　　要：为制备新型鸭源呼肠孤病毒(DRV)TH11株Sigma A蛋白的多克隆抗体,本研究采用RT-PCR方法扩增DRV TH11株Sigma A的编码基因,将其克隆至原核表达载体pET-32a(+)中,在大肠杆菌BL21(DE3)中进行诱导表达。采用SDS-PAGE和western blot对表达产物进行鉴定。结果表明,Sigma A基因不仅可以在大肠杆菌中高水平表达,表达产物的分子量约66 ku,而且表达产物能够被特异性DRV多克隆抗体识别,证明表达的Sigma A蛋白具有良好的免疫活性。以纯化的Sigma A蛋白免疫实验兔制备抗Sigma A蛋白的多克隆抗体,间接ELISA检测结果显示其效价达1∶20 000以上。间接免疫荧光试验表明,多克隆抗体能够特异性识别DRV的Sigma A蛋白,表明Sigma A蛋白具有良好的免疫原性。本研究为进一步研究Sigma A蛋白的功能,以及建立DRV检测方法奠定了基础。To prepare polyclonal antibody against Sigma A protein of a novel duck reovirus （DRV）, the Sigma A gene was amplified by RT-PCR and cloned into the pET-32a（＋） vector. The resultant recombinant plasmid was transformed into E.coli BL21 （DE3） to express with IPTG induction. The SDS-PAGE analysis showed that the expressed product was about 66ku which was recognized by antiserum of DRV TH-11 reovirus by western blot. The polyclonal antibody was prepared from the rabbit immunized with purified recombinant protein. The titer of the antibody was about 1：20,000 by detection of indirect ELISA. The indirect immunofluorescence assay also confirmed that the polyclonal antibody reacted specially with DRV Sigma A protein, which would be used for study of the Sigma A function.

关 键 词：鸭源呼肠孤病毒 SIGMA A基因 原核表达 免疫原 

分 类 号：S852.65[农业科学—基础兽医学]