牛病毒性腹泻病毒E2结构蛋白的原核表达、纯化及鉴定

根据GenBank中已发表的牛病毒性腹泻病毒囊膜糖蛋白E2基因设计1对特异性引物,以Oregon CV24株为模板,PCR扩增目的片断后与pET-32a原核表达载体连接,转化大肠杆菌Transetta（DE3）感受态细胞,用IPTG诱导表达,SDS-PAGE电泳分析表达产物,结果表明重组E2蛋白在体外得到较好表达,表达的融合蛋白分子量为57.7 ku。可溶性分析表明重组E2蛋白以包涵体的形式存在,经镍柱His标签蛋白纯化后进行western-blot检测。结果表明：E2重组蛋白均具有反应原性,能与BVDV的阳性血清进行反应。

Expression and Identification of the Structural Protein E2 of Bovine Virus Diarrhea

A pair of primers was designed according to the major antigenic region of bovine virus diarrhea E2 gene derived form GenBank, and was used to amplify the E2 gene of Oregon CV24 strain by PCR method. The E2 gene was subcloned into prokaryotic vector Pet-32a to construct recombinant expression plasmid. The recombinant plasmid was transformed into Escherichia coli Transetta（DE3）, and then induced by IPTG. The E2 recombinant protein was analyzed by SDS-PAGE and western blot. The result showed that the E2 recombinant protein was mainly expressed in the form of inclusion body and the molecular weight of the fusion protein was about 57.7 ku. The result of the western-blot showed that the E2 recombinant protein could be specifically recognized by BVDV positive serum, and may be used as the coating antigen to develop a diagnosis kit against BVDV.