禽戊型肝炎病毒CaHEV-GDSZ01株ORF3蛋白的原核表达及其多克隆抗体的制备

Prokaryotic Expression and Polyclonal Antibody Preparation of ORF3 Protein of Avian hepatitis E virus CaHEV-GDSZ01 Strain

旨在对禽戊型肝炎病毒的ORF3蛋白进行原核表达,并制备其多克隆抗体,为进一步研究aHEV ORF3蛋白生物学功能新型疫苗提供生物材料。通过RT-PCR方法扩增CaHEV-GDSZ01株ORF3基因,连接pET-32a(+)表达载体以构建重组原核表达质粒,并转化BL21(DE3)感受态细胞进行诱导表达;利用SDS-PAGE凝胶电泳分析ORF3蛋白的表达形式后进行纯化,并免疫新西兰白兔以获得兔抗ORF3蛋白多克隆抗体,利用间接ELISA试验检测多克隆抗体的效价,利用Western Blot试验和间接免疫荧光试验(IFA)检测多克隆抗体特异性。结果显示,成功构建了pET-32a-ORF3重组原核表达质粒,并通过原核表达系统成功诱导表达了27 ku不可溶性的aHEV ORF3重组蛋白;Western Blot试验和IFA试验结果显示,制备的多克隆抗体可以特异性识别ORF3蛋白,ELISA结果显示,多克隆抗体效价为1∶51 200。成功表达CaHEV-GDSZ01株的ORF3蛋白,并制备了其多克隆抗体。

The aim of this study was to express the ORF3 protein of Avian hepatitis E virus(aHEV)and prepare its polyclonal antibodies,which can provide biomaterials for further study on the biological function of new vaccine of aHEV ORF3 protein.The ORF3 gene of CaHEV-GDSZ01 strain was amplified by RT-PCR,ligated with pET-32a(+)expression vector to construct recombinant prokaryotic expression plasmid,and transformed into BL21(DE3)competent cells to induce expression.The expression of ORF3 protein was analyzed by SDS-PAGE gel electrophoresis and immunization of rabbits to obtain rabbit anti-ORF3 polyclonal antibody.The titer of polyclonal antibody was determined by indirect ELISA,and its specificity was detected by Western Blot and IFA.The pET-32a-ORF3 recombinant prokaryotic expression plasmid was successfully constructed,and the 27 ku insoluble recombinant protein of aHEV ORF3 was successfully expressed by prokaryotic expression system.The results of Western Blot and IFA showed that the prepared polyclonal antibody could specifically recognize ORF3 protein,and the ELISA results showed that the titer of polyclonal antibody was 1∶51200.In this study,the ORF3 protein of CaHEV-GDSZ01 strain was successfully expressed and its polyclonal antibody was prepared.