论文部分内容阅读
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。
OBJECTIVE: To prokaryotic express and purify VP0 protein of human enterovirus 71 (EV71) and to immunize guinea pigs to prepare polyclonal antibodies and to identify their reactivity and specificity for the detection of EV71. Methods: The VP0 gene was amplified by PCR. The prokaryotic expression plasmid pET-VP0 was constructed and transformed into E.coli. VP0 recombinant protein was induced and purified. The anti-VP0 polyclonal antibody was prepared by immunization of guinea pigs. The anti-VP0 antibody titer was detected by ELISA. And Western blot to identify antibody specificity. Results: VP0 recombinant protein was highly expressed in E. coli BL21. The anti-VP0 antibody titer was 1:10. Immunofluorescence and Western blot results showed that anti-VP0 polyclonal antibody could recognize prokaryotic expression and VP0 protein in EV71 infected cells. Conclusion: The prokaryotic expression of VP0 protein of EV71 and the preparation of polyclonal antibodies against VP0 provide new research tools and tools for the clinical diagnosis, vaccine development and molecular virology of EV71.