A型塞内卡病毒VP3蛋白的原核表达及其多克隆抗体的制备

Prokaryotic expression of VP3 protein of Senecavirus A and preparation of its polyclonal antibody

为了研究A型塞内卡病毒(Senecavirus A,SVA)VP3蛋白的功能,将SVA的VP3基因克隆至原核表达载体pET-32a(+)构建重组质粒p ET32a-SVA-VP3,转化大肠杆菌BL21(DE3)后进行诱导表达;用纯化复性后的重组蛋白免疫新西兰大白兔制备多克隆抗体,并用Western-blot与间接免疫荧光试验鉴定其特异性。结果表明,重组SVA VP3蛋白在大肠杆菌BL21(DE3)以包涵体形式表达,分子质量约为44 ku;制备的家兔多克隆抗体能与SVA表达的VP3蛋白特异性结合,而不与口蹄疫病毒抗原反应,表明制备的SVA VP3蛋白家兔多抗血清具有良好的反应原性和特异性。重组SVA VP3蛋白及其多克隆抗体的成功制备为SVA血清学检测方法的建立、SVA致病机制及VP3蛋白功能的研究提供了材料支撑。

To study the function of Senecavirus A(SVA)VP3 protein,VP3 gene sequence of SVA was cloned into the prokaryotic expression vector p ET-32a(+)to construct recombinant plasmid p ET32a-SVAVP3,which was transformed into Escherichia coli BL21(DE3)cells to induce the expression of recombinant protein.Polyclonal antibody was prepared by immunizing New Zealand white rabbits with purified and renaturated recombinant VP3 protein,and the specificity of polyclonal antibody was determined by Western-blot and indirect immunofluorescence assay.The results showed that the recombinant protein SVA VP3 was expressed in E.coli BL21(DE3)cells in the form of inclusion bodies with a molecular weight of 44 ku.The prepared rabbit polyclonal antibody can specifically bind to VP3 protein expressed by SVA,but not react with foot-and-mouth disease virus antigen,indicating that the prepared rabbit polyclonal antibody had good reactivity and specificity.The successful preparations of recombinant SVA VP3protein and its polyclonal antibody provided material support for the establishment of serological detection method of SVA,the study of the pathogenesis of SVA and the function of VP3 protein.