基于荧光素酶报告基因的细菌外膜囊泡标记方法的建立

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。

Establishment of bacterial outer membrane vesicle labeling method based on luciferase reporting system

Objective To establish a bacterial outer membrane vesicle labeling method based on luciferase reporting system. Methods Using the characteristics of high abundance outer membrane protein on the surface of bacterial outer membrane vesicles, the outer membrane protein-luciferase fusion protein was constructed to realize the " Self-luminescent" of bacterial outer membrane vesicles for detection. Results The luciferase enzyme markers of bacterial outer membrane vesicles were constructed by constructing OmpA-NanoLuc fusion protein, and the outer membrane vesicles secreted by bacteria expressing the fusion protein had definite luciferase activity, the bacterial outer membrane vesicles could be detected by detecting the activity of fluorescein enzyme. Conclusion A bacterial outer membrane vesicle labeling method based on fluorescein enzyme reporting gene was successfully constructed, and the bacterial outer membrane vesicles could be detected without the use of cell membrane dyes.