【摘要】 目的 原核表达肠道病毒71型3C蛋白酶（enterovirus 71 3C protease, EV71-3Cpro）并制备其特异性多克隆抗体。方法 首先依据密码子优化原则合成EV71-3Cpro基因，将其克隆到pET-28a表达载体NdeⅠ和XhoⅠ位点间构建重组质粒，再以冷CaCl₂法将其转化到大肠杆菌E.coli Rosetta（DE3）感受态细胞中，优化原核表达条件。以HisTrap^TM亲和层析柱进行分离纯化，以纯化的EV71-3Cpro作为抗原免疫大鼠，分离血清后以酶联免疫吸附实验（enzyme-linked immunosorbent assay, ELISA）、蛋白质印迹法（western blotting, WB）鉴定多克隆抗体的效价及其特异性。结果 ELISA和WB结果显示制备的大鼠抗EV71-3Cpro多克隆抗体效价达1∶1 024 000,且具有较好的特异性。结论 抗EV71-3Cpro多克隆抗体的成功制备为EV71-3Cpro生物学功能的研究及其联合疫苗的开发奠定了实验基础。更多还原

【Abstract】 Objective To cultivate enterovirus 71 3C protease（EV71-3Cpro）in prokaryotic cells with prokaryotic expression and prepare its specific polyclonal antibody. Methods Firstly, EV71-3Cpro gene was synthesized according to codon optimization principle, cloned into pET-28a expression vectors NdeⅠ and XhoⅠ to construct recombinant plasmid, and then transformed into E.coli Rosetta（DE3）receptor cells by cold CaCl₂ method to optimize prokaryotic expression conditions. HisTrapTM affinity chromatography was used to isolate and purify EV71-3Cpro, and the purified EV71-3CPro was used as antigen to immunize rats. Enzym-linked immunosorbent assay（ELISA）and Western Blotting（WB）were used to evaluate the titer and specificity of the polyclonal antibody after separating the serum. Results ELISA and WB results showed that the prepared rat anti-EV71-3CPro polyclonal antibody had a titer of 1∶1 024 000,and had good specificity. Conclusion The successful preparation of the anti-EV71-3Cpro polyclonal antibody laid the experimental foundation for the study of the biological function of EV71-3Cpro and the development of the combined vaccine.更多还原