人肠道病毒71型VP0蛋白的原核表达及多克隆抗体制备

Expression of VP0 protein of enterovirus 71 in Escherichia coli and generation of the corresponding polyclonal antibodies in guinea pigs

目的:原核表达并纯化人肠道病毒71型(EV71)VP0蛋白,免疫豚鼠制备多克隆抗体,并鉴定其用于EV71检测的反应性和特异性。方法:PCR方法扩增VP0基因,构建原核表达质粒pET-VP0并转化大肠杆菌,诱导表达并纯化VP0重组蛋白,免疫豚鼠制备抗VP0多克隆抗体,ELISA方法检测抗VP0抗体效价,细胞免疫荧光和Western blot方法鉴定抗体特异性。结果:VP0重组蛋白在大肠杆菌BL21中高效表达,制备的抗VP0抗体效价为1∶106。细胞免疫荧光及Western blot检测结果显示,抗VP0多克隆抗体可以识别原核表达及EV71感染细胞中的VP0蛋白。结论:原核表达了EV71的VP0蛋白并制备出抗VP0多克隆抗体,为EV71的临床诊断、疫苗开发和分子病毒学研究提供了新的研究工具和手段。

AIM： To obtain recombinant VP0 protein of enterovirus 71,and generate the corresponding VP0-specific polyclonal antibodies,for molecular detection and characterization of EV71.METHODS： The VP0 gene was amplified by PCR and cloned into vector pET26b to make pET-VP0 for the prokaryotic expression of VP0.The recombinant VP0 protein was expressed in E.coli BL21 harboring pET-VP0,purified from inclusion bodies,renatured,and subsequently used to immunize guinea pigs.The resultant antisera were evaluated for anti-VP0 titer,binding capacity and specificity by ELISA,immunofluorescence staining and Western blot assays.RESULTS： Recombinant protein VP0 was efficiently produced in E.coli.Immunization of guinea pigs with recombinant VP0 elicited high-titer（1∶106） VP0-specific antibodies.Western blot analysis showed the resultant anti-VP0 sera reacted with E.coli-expressed VP0 as well as EV71 propagated in Vero cells.Moreover,the antisera positively recognized EV71 infected cells by immunofluorescence staining.CONCLUSION： The recombinant VP0 and the corresponding polyclonal antibodies can be used to identify and characterize EV71,and therefore represent useful agents and tools for the development of new diagnostic methods and vaccines for EV71.