摘要
目的建立一种基于荧光素酶报告基因的细菌外膜囊泡标记方法。方法利用细菌外膜囊泡表面携带高丰度细菌外膜蛋白A(OmpA)的特点,构建OmpA-NanoLuc荧光素酶融合蛋白,根据荧光素酶活性评估样品中的细菌外膜囊泡数量。结果成功构建OmpA-NanoLuc融合蛋白表达载体,受试菌株在表达该融合蛋白后所分泌的外膜囊泡具有稳定的荧光素酶活性。可通过检测荧光素酶活性的方式对细菌外膜囊泡进行半定量检测。结论建立了一种基于荧光素酶标记的细菌外膜囊泡半定量检测方法,可用于评估特定菌株的细胞外膜囊泡分泌水平。
Objective To establish a labeling method for bacterial outer membrane vesicle(OMV)based on luciferase reporting gene.Methods By utilizing the characteristics that high abundance of outer membrane protein A(OmpA)presented on the surface of bacterial OMV,the outer membrane protein-luciferase fusion protein was constructed to position the luciferase on the surface of the outer membrane vesicle,and the number of bacterial OMV was evaluated based on the luciferase activity.Results The OmpA-NanoLuc fusion protein expression vector was constructed successfully,and the outer membrane vesicles secreted by the subject strains after the expression of the fusion protein displayed stable luciferase activity.The number of bacterial outer membrane vesicles was semi-quantitative detrmined by measuring the activity of fluorescein enzyme.Conclusion A semi-quantitative method based on luciferase labeling was developed for the detection of extracellular vesicles,which could be used to evaluate the secretion level of specific strains.
作者
刘畅
李楚楚
李智洋
LIU Chang;LI Chuchu;LI Zhiyang(Department of Laboratory Medicine,Nanjing Drum Tower Hospital,Nanjing 210008,Jiangsu;Department of Acute Infectious Disease Control and Prevention,Jiangsu Provincial Center for Disease Control and Prevention,Nanjing 210009,Jiangsu,China)
出处
《临床检验杂志》
CAS
2019年第10期742-744,共3页
Chinese Journal of Clinical Laboratory Science
基金
南京市卫生科技发展专项资金(YKK18066)
中国博士后基金第65批面上资助(2019M651802)
江苏省干部保健科研课题(BJ16003)
南京市科技发展计划项目(201715020)
关键词
细菌外膜囊泡
外膜蛋白
荧光素酶
bacterial outer membrane vesicles
outer membrane protein
luciferase
