摘要目的 利用枯草芽孢杆菌系统表达肠道病毒71型(EV71)病毒VP1蛋白.方法 使用基因特异性引物扩增VP1开放读码框(ORF),构建包含VP1完整开放读码框的pSac-VP1穿梭载体.根据枯草芽孢杆菌表达系统双交叉同源重组的特点,将EV71的结构抗原基因VP1整合在枯草芽孢杆菌sacA染色体,并经测序鉴定.使用VP1特异性抗体,通过蛋白印记(Western-Blot)鉴定枯草芽孢杆菌中VP1蛋白的表达情况.结果 成功克隆EV71的VP1基因,长度1361 bp,并将其克隆入穿梭载体pSac-Kan.测序结果显示VP1基因成功整合如sacA染色体.Western-Blot证实VP1抗原在枯草芽孢杆菌的成功表达,相对分子质量约35×103.结论 利用枯草芽孢杆菌表达系统成功制备EV71病毒VP1抗原,为后续EV71诊断试剂、疫苗研发奠定基础.

abstractsObjective To express the recombinant VP1 protein of enterovirus 71 in bacillus subtilis expression system.Methods We first amplified ORF of VP1 gene with specific primer and constructed pSac-VP1 vector.According to the characteristic of homologous recombination in bacillus subtilis,we integrated EV71 VP1 gene into bacillus subtilis sacA chromosome identified with sequencing.VP1 protein expression was identified with Western-Blot assay using specific antibody.Results VP1 gene fragment was amplified successfully with 1361 bp and inserted into pSac-Kan vector.After homologous recombination,the VP1 gene was integrated into bacillus subtilis sacA chromosome.It was proved that the recombinant VP1 proteins could be expressed in bacillus subtilis with molecular weight about 35 × 103.Conclusion We expressed recombinant VP1 of EV71 in bacillus subtilis system,it would benefit the diagnosis and vaccine research of EV71 in the future.