摘要
为了对猪伪狂犬病病毒(Pseudorabies virus,PRV)EP0蛋白进行原核表达并制备其多克隆抗体,试验以PRV HeN1毒株为模板,构建了EP0基因原核重组质粒pET28a-EP0,经测序鉴定后,将pET28a-EP0重组质粒转化至大肠杆菌BL21(DE3)感受态细胞中以表达重组蛋白His-EP0;确认重组蛋白His-EP0表达后,用Ni-NTA柱纯化重组蛋白,并用纯化的重组蛋白免疫Balb/c小鼠制备多克隆抗体,进行Western-blot测定,用间接ELISA法测定多克隆抗体效价,用间接免疫荧光法测定多克隆抗体的特异性。结果表明:EP0蛋白原核表达成功,His-EP0蛋白在大肠杆菌BL21(DE3)感受态细胞中以可溶性蛋白形式表达,大小在55~70 ku之间;EP0蛋白多克隆抗体制备成功,抗体效价在1∶160000以上,且能与表达的重组蛋白His-EP0及PRV感染的细胞发生特异性反应。说明试验成功表达了EP0蛋白,并成功制备了EP0蛋白多克隆抗体,抗体效价可达到1∶160000以上,且特异性较好。
To express EP0 protein of Pseudorabies virus(PRV)and prepare polyclonal antibody,the EP0 prokaryotic recombinant plasmid pET28a-EP0 was constructedby using PRV HeN1 strain as template.After sequencing identification,pET28a-EP0 recombinant plasmid was transformed into the Escherichia coli(E.coli)BL21(DE3)competent cells to express recombinant protein His-EP0.After confirming the expression of recombinant protein His-EP0,the recombinant protein was purified by Ni-NTA column and its immunized Balb/c mice were used it to prepare polyclonal antibodies.The preparation of polyclonal antibody was determined by Western-blot,the titer of polyclonal antibody was determined by indirect ELISA,and the specificity of polyclonal antibody was determined by indirect immunofluorescence.The results showed that EP0 protein was successfully expressed in prokaryotic form,and His-EP0 protein was expressed as a soluble protein in E.coli BL21(DE3)competent cells with a size ranging from 55 ku to 70 ku.Polyclonal antibody against EP0 protein was successfully prepared.The titer of the antibody was over 1∶1600000,and it could react specifically with the expressed recombinant protein His-EP0 and PRV infected cells.The results indicated that EP0 protein was successfully expressed and polyclonal antibody against EP0 protein was successfully prepared.The titer of antibody was over 1∶1600000 and the specificity was good.
作者
赵鸿远
马宁宁
王迪
ZHAO Hongyuan;MA Ningning;WANG Di(School of Modern Agriculture&Biotechnology,Ankang University,Ankang 725000;School of Agroforestry and Medicine,Open University of China,Beijing 100093,China)
出处
《黑龙江畜牧兽医》
CAS
北大核心
2022年第15期18-22,136,共6页
Heilongjiang Animal Science And veterinary Medicine
基金
国家自然科学基金项目(31902302)
安康学院科技项目(2019AYQJ11)
安康学院科技创新团队项目(2022TD02)大学生创新创业训练计划项目(s201911397060)。
