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目的利用昆虫杆状病毒表达系统构建肠道病毒71型及柯萨奇病毒A16型(EV71/CA16)联合P1基因(HP1)重组杆状病毒。方法利用重叠PCR的原理,设计合成HP1基因;HP1基因与载体pFastBac1连接后转化DH10Bac感受态细胞,通过蓝白斑筛选获得重组穿梭杆粒HP1-bacmid;提取纯化重组杆粒转染Sf9细胞,收获重组杆状病毒;以SDS-PAGE、Western blot进行重组蛋白鉴定。结果成功合成HP1基因;完成HP1与pFastBac1载体连接;获得了重组穿梭杆粒HP1-bacmid;并通过转染Sf9细胞,获得具有感染活性的重组杆状病毒;SDS-PAGE电泳结果显示重组病毒感染的Sf9细胞表达了大小约为100×103 Mr的目的蛋白条带,经Western blot鉴定,该条带能与EV71单克隆抗体特异结合。结论成功构建了EV71/CA16联合P1基因重组杆状病毒,为下一步EV71/CA16联合P1蛋白的免疫鉴定奠定基础,并为兼抗EV71、CA16疫苗的研究提供参考。
Objective To construct enterovirus 71 and coxsackievirus A16 (EV71 / CA16) combined with P1 gene (HP1) recombinant baculovirus by using insect baculovirus expression system. Methods The HP1 gene was designed and synthesized based on the principle of overlap PCR. After the HP1 gene was ligated with the vector pFastBac1, the recombinant plasmid was transformed into DH10Bac competent cells. The recombinant shuttle plasmid HP1-bacmid was obtained by blue-white screening. The purified recombinant bacmid was transfected into Sf9 cells, Baculovirus; SDS-PAGE, Western blot recombinant protein identification. RESULTS: HP1 gene was successfully synthesized. HP1 was ligated with pFastBac1 vector. The recombinant shuttle bacmid HP1-bacmid was obtained. The recombinant baculovirus was transfected into Sf9 cells. The results of SDS-PAGE showed that recombinant HP1- Sf9 cells expressed a size of about 100 × 103 Mr protein band, identified by Western blot, the band with EV71 monoclonal antibody specific binding. Conclusion The recombinant baculovirus EV71 / CA16 combined with P1 gene was successfully constructed, which laid the foundation for the next immunization identification of EV71 / CA16 combined with P1 protein and provided a reference for the study of EV71 and CA16 vaccines.