应用枯草芽孢杆菌表达系统制备肠道病毒71型VP1蛋白  

The expression of enterovirus 71 VP1 gene with bacillus subtilis system

作　　者：张国良[1] 刘威龙[1] 詹森林[1] 刘映霞[1] 陈圆圆[1] 杨桂林[1] 聂广[1] 周伯平[1] 

机构地区：[1]深圳市第三人民医院,518112

出　　处：《中华实验和临床病毒学杂志》2013年第6期403-405,共3页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金（30972603）;深圳市科技计划项目（CXB201104250061A）

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VPl蛋白。方法使用基因特异性引物扩增VPl开放读码框（ORF），构建包含VP1完整开放读码框的pSac—VPl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VPl整合在枯草芽孢杆菌sacA染色体，并经测序鉴定。使用VPl特异性抗体，通过蛋白印记（Western—Blot）鉴定枯草芽孢杆菌中VPl蛋白的表达情况。结果成功克隆EV71的VPl基因，长度1361bp，并将其克隆入穿梭载体pSae—Kan。测序结果显示VPl基因成功整合如sacA染色体。Western．Blot证实VPl抗原在枯草芽孢杆菌的成功表达，相对分子质量约35×10^3。结论利用枯草芽孢杆菌表达系统成功制备EV71病毒VPl抗原，为后续EV71诊断试剂、疫苗研发奠定基础。Objective 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 x 103. Conclusion We expressed recombinant VP1 of EVT1 in bacillus subtilis system,it would benefit the diagnosis and vaccine research of EV71 in the future.

关 键 词：肠道病毒属 病毒包膜蛋白质类 芽孢杆菌 枯草 

分 类 号：R373[医药卫生—病原生物学]