摘要目的 原核表达肠道病毒68型(enterovirus D68,EV-D68)表面结构蛋白VP2、VP3,分析其亲和力和结合力,得到具备高结合力的、可结合特异性记忆性B细胞的EV-D68病毒表面结构蛋白.方法 采用PCR法扩增EV-D68病毒的VP2、VP3蛋白基因,插入原核表达载体pGEX-5X-1,构建重组表达质粒pGEX-5X-1-VP2和pGEX-5X-1-VP3,转化到工程菌Rosetta(DE3)pLy中,异丙基硫代半乳糖苷(i-sopropylβ-D-thiogalactoside,IPTG)诱导表达,表达的重组蛋白形成包涵体,经超声洗涤、透析复性;采用聚丙烯酰胺凝胶电泳(polyacrylamide gel electrophoresis,SDS-PAGE)电泳、Western-blot鉴定重组蛋白;竞争性酶联免疫吸附检测(enzyme linked immunosorbent assay,ELISA)检测蛋白VP2、VP3的蛋白活性和亲和力;藻红蛋白荧光素(phycoerythrin,PE)标记VP2和VP3重组蛋白,结合CD19-APC、CD27-FITC抗体,流式细胞术检测VP2、VP3蛋白结合特异性记忆性B细胞的情况.结果 成功构建出重组表达质粒pGEX-5X-1-VP2和pGEX-5X-1-VP3,通过双酶切鉴定及测序分析,确认表达载体构建正确,通过表达纯化,成功获得EV-D68的VP2、VP3蛋白,SDS-PAGE电泳灰度扫描显示,VP2蛋白的纯度可达80％,VP3蛋白的纯度可达75％;经Western-blot和ELISA鉴定,均有抗原特异性;通过竞争ELISA检测,VP2亲和力常数为2.7×107 M-1,为中等亲和力;VP3亲和力常数为3.25×106 M-1,为低亲和力;通过流式细胞术可以检测到病毒攻击小鼠后脾脏细胞中的特异性记忆性B细胞.结论 成功原核表达,复性纯化得到EV-D68 VP2、EV-D68 VP3蛋白,两个蛋白均具有较强的抗原特异性,利用流式细胞仪能检测到特异性记忆性B细胞.最终获得了具有较强结合能力,可用于分选抗原特异性记忆性B细胞的EV-D68表面结构蛋白VP2、VP3,为研究VP2、VP3蛋白的中和抗原表位及其广谱中和抗体的分选检测打下了基础.

abstractsObjective VP2 and VP3 surface structural proteins of EV-D68 were expressed in prokary-otic system,and the affinity and binding charactors of two proteins were analyzed to investigate their capacity of binding to memory B cells. Methods The VP2 and VP3 genes of EV-D68 were amplified by PCR and inserted into prokaryotic expression vector pGEX-5X-1. Rosetta(DE3)pLysS,transformed by the constructed recombinant expression plasmids pGEX-5X-1-VP2 and pGEX-5X-1-VP3,were induced by IPTG. The expressed recombinant protein in the form of inclusion bodies were extracted,followed by washing,dialyzing and renaturing. The recom-binant protein was then identified by SDS-PAGE electrophoresis and Western blotting. The activity and affinity of VP2 and VP3 were evaluated by competitive ELISA. The binding capacity of VP2 and VP3 to the memory B cells was detected by flow cytometry. Results The recombinant expression plasmids pGEX-5X-1-VP2 and pGEX-5X-1-VP3 were successfully constructed and verified by double restriction enzyme digestion and sequen-cing. We then obtained both VP2 and VP3 proteins. The purity of VP2 and VP3 proteins were 80％ and 75％, respectively. Both proteins showed antigenic specificity. VP2 showed a medium affinity with affinity constant of 2. 7 × 107 M - 1 ,and the VP3 showed a low affinity with affinity constant of 3. 25 × 106 M - 1 . Specific memory B cells in spleen after viral challenge to mice can be detected by flow cytometry. Conclusion VP2 and VP3 proteins of EV-D68 were obtained in prokaryotic system with favorable antigen specificity. These proteins can specifically bind to memory B cells with high affinity and binding capacity. These findings offer the targets for sorting EV-68-specific memory B cellsand provide insight for further investigation in the neutralizing epitopes of VP2 and VP3 and their broad-spectrum neutralizing antibodies.