日本脑炎病毒E III结构域蛋白的表达及多克隆抗体的制备与鉴定

摘要

使用原核表达系统融合表达乙型脑炎病毒EIII结构域蛋白，经过纯化后多次免疫BALB/c小鼠，制备了鼠抗JEV E蛋白的多克隆抗体。Western blot结果表明该抗体与重组蛋白能够发生明显反应，与浓缩的JEV反应显出1条分子量大小约为53 kDa的目的条带（JEV E蛋白）；间接免疫荧光（indirect immunofluorescence，IFA）显示，多克隆抗体与BHK-21细胞内的JEV作用，在细胞质内产生绿色荧光，而对照组无特异性荧光；空斑减少中和试验测定多抗血清的中和抗体效价（PRNT50）为1：160。以上结果暗示制备的多克隆抗体能与重组蛋白和JEV E蛋白都能发生良好反应，并且该多抗有较高水平的JEV中和抗体效价。%In this study, E protein domain III (EDIII) of JEV was expressed in E.coli and purified with a His column. BALB/c mice were immunized with EDIII and polyclonal antibodies to E protein were prepared. The reactivity of the prepared antibodies was characterized in Western blotting and IFA. Furthermore, the polyclonal antibodies at dilution of 1:160 blocked the entry of JEV into BHK-21 cells in plaque reduction neutralization test. These results demonstrated that the polyclonal antibodies of JEV EDIII possessed good antigenic specificity and could be used for development of a diagnostic method of JEV infection.