猪流行性腹泻病毒S蛋白的原核表达及其多克隆抗体的制备

Prokaryotic expression and polyclonal antibody preparation of spike protein of porcine epidemic diarrhea virus

为深入研究猪流行性腹泻病毒(PEDV)的结构和功能及建立PEDV的血清学检测方法,运用基因克隆技术将目的基因克隆至表达载体pET-30a(+)中,将重组质粒转化至大肠杆菌BL21(DE3)进行诱导表达,表达产物纯化后免疫新西兰大白兔获得抗S蛋白的多克隆抗体。SDS-PAGE试验结果显示,成功表达大小为14.2 ku和47.5 ku的目的重组蛋白,Western-blot试验显示目的条带与预期相符且条带单一。对获得的多克隆抗体进行Western-blot和间接免疫荧光验证的结果显示,PVDF膜上的条带与预期条带一致且感染PEDV的Vero细胞内出现特异性绿色荧光信号。结论,本研究通过表达纯化PEDV S蛋白,成功制备具有良好免疫原性的家兔多克隆抗体,为深入研究PEDV的结构和功能奠定了基础,为PEDV的血清学检测方法的建立提供了生物学材料。

In order to investigate the structure and function of porcine epidemic diarrhea virus(PEDV)and to establish a serological assay for PEDV,the target genes were cloned into the expression vector p ET-30a(+),and the recombinant plasmids were transformed into Escherichia coli BL21(DE3)to induce expression.The polyclonal antibodies against S protein were obtained by immunizing New Zealand white rabbits.SDS-PAGE assay showed that the target recombinant proteins with the size of 14.2 and47.5 ku were successfully expressed,and Western-blot test showed that the target protein bands were consistent with the expected and the bands were single.Western-blot and indirect immunofluorescence used the obtained polyclonal antibodies showed that the bands on the PVDF membrane were consistent with the expected and there were specific green fluorescence signal in PEDV-infected Vero cells.In conclusion,the rabbit polyclonal antibodies with good immunogenicity were successfully prepared by expressing and purifying PEDVS protein,which laid a foundation for further study of the structure and function of PEDV and provided biological materials for the establishment of serological detection of PEDV.