摘要
目的比较不同实验条件下SpyTag/SpyCatcher体系共价连接的效率,分析得出优化后的连接条件,为今后相关纳米颗粒疫苗的开发提供实验依据。方法用大肠埃希菌表达分别带有SpyTag和SpyCatcher结构的两种蛋白,摸索共价连接的反应时间、温度、缓冲体系和混合比例,采用非还原型凝胶电泳检测共价连接的效率。结果反应时间对共价连接的影响有统计学意义(F=22.028,P<0.05),带有SpyTag和SpyCatcher结构的两种蛋白质可形成稳定的共价键,6 h后连接效率约为50%,偶联蛋白在25℃稳定存在至少72 h。反应温度和缓冲体系对共价连接的影响没有统计学意义。SpyTag和SpyCatcher结构蛋白比例为物质的量比1.0∶2.0混合。结论SpyTag/SpyCatcher共价连接体系的关键工艺参数为反应时间,且该体系能适用于多种温度和缓冲条件。
Objective To investigate the covalent bonding efficiency of SpyTag/SpyCatcher system under different experimental conditions.Methods Two proteins with the structure of SpyTag and SpyCatcher respectively were expressed in E.coli.To detect the efficiency of covalent bonding by non-reducing gel electrophoresis when the reaction time,temperature,buffer system,and mixing ratio were changed.Results The reaction time had a significant effect on covalent bonding(F=22.028,P<0.05).When SpyTag and SpyCatcher were mixed,covalent bonding occurred immediately,the bonding efficiency reached about 50%after 6 h,and the new protein was stable at 25℃for at least 72 h.The reaction temperature and buffer system had no significant effect on covalent bonding.In this study,the appropriate mixing molar ratio of the two proteins was 1.0∶2.0.Conclusion The critical process parameter of SpyTag/SpyCatcher covalent bonding system is reaction time,and this system can be applied to a variety of temperature and buffer conditions.
作者
王蓓
胡海涛
楼觉人
Wang Bei;Hu Haitao;Lou Jueren(Research and Development Department,Shanghai Wellvax Co.,Ltd.,Shanghai 200051,China;No.2 Laboratory,Shanghai Institute of Biological Products Co.,Ltd.,Shanghai 200051,China)
出处
《国际生物制品学杂志》
CAS
2021年第5期271-275,共5页
International Journal of Biologicals
