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 用大肠埃希菌系统表达分别带有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. The efficiency of covalent bonding was detected 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.