水牛匈爱病毒结构蛋白VP1的截短表达及多克隆抗体的制备

Truncated expression of structural protein VP1 of Water buffalo Hunnivirus and preparation of polyclonal antibody

为了制备水牛匈爱病毒(BufHuV)结构蛋白VP1截短基因的多克隆抗体,将病毒的VP1截短基因克隆至原核表达载体pET-32a(+)获得重组质粒pET-32a-BufHuV-VP1,重组质粒转化至BL21感受态细胞(DE3)后诱导表达,VP1截短蛋白的分子质量约为31.5 ku,主要以包涵体的形式表达。以纯化好的重组蛋白免疫昆明小鼠以制备多克隆抗体,通过Western-blot与IFA鉴定其特异性。结果显示,制备的小鼠多克隆抗体能与纯化的VP1重组截短蛋白和感染BufHuV的细胞表达的VP1特异性结合,表明多克隆抗体具有良好的反应原性和特异性。水牛匈爱病毒VP1截短重组蛋白及其多克隆抗体的成功制备为研究BufHuV致病机制、VP1蛋白功能以及血清学检测方法的建立提供了材料基础。

In order to prepare polyclonal antibody against truncated VP1 gene of Water buffalo Hunnivirus(BufHuV)structural protein,,the truncated VP1 gene of BufHuV was cloned into prokaryotic expression vector pET-32a(+)and the recombinant plasmid pET-32a-BufHuV-VP1 was obtained.The recombinant plasmid was transformed into BL21(DE3)to induce expression.The molecular weight of the truncated VP1 protein was about 31.5 ku,and it was mainly expressed in the form of inclusion body.Kunming mice were immunized with the purified recombinant protein to prepare polyclonal antibody,and its specificity was identified by Western-blot and IFA.The results showed that the mouse polyclonal antibody could bind to the purified VP1 recombinant truncated protein and the VP1 expressed in BufHuV-infected cells,which indicated that the polyclonal antibody had good reactivity and specificity.The successful preparation of truncated recombinant VP1 protein and its polyclonal antibody of BufHuV provides a material basis for the study of pathogenesis,function of VP1 protein and establishment of serological detection method of BufHuV.