空肠弯曲菌双分子荧光互补系统构建及其在蛋白质互作研究中的应用

Construction of a bimolecular fluorescence complementation system for Campylobacter jejuni and its application in protein interaction studies

为研究空肠弯曲菌中蛋白质的相互作用,建立适合于该菌稳定高效表达的双分子荧光互补(bimolecular fluorescence complementation,BiFC)系统,通过构建基于基因启动子、荧光蛋白以及基因回补方式等不同条件的重组载体,分别导入空肠弯曲菌进行荧光数值比较分析,确定荧光表达最优的参数,建立以porA启动子和Venus荧光蛋白的质粒回补的BiFC体系。用该体系探究空肠弯曲菌鞭毛表达蛋白的互作。结果表明:已知flhF与flhG互作的BiFC-flhF-flhG载体在菌体内表达的荧光值显著高于阴性对照载体,而BiFC-flhF-fliM和BiFC-flhF-fliG与阴性对照无差异,说明BiFC体系建立成功,且flhF与fliG、fliM不存在互作关系。综上,本研究建立了适用于空肠弯曲菌的BiFC体系,可为该菌蛋白质互作研究提供高效便捷的方法。

In order to study protein interactions in C.jejuni,a bimolecular fluorescence complementation(BiFC)system suitable for stable and efficient expression in this bacterium was established.By constructing recombinant vectors based on different conditions of gene promoters,fluorescent proteins,and gene complementation,respectively,and introducing them into C.jejuni for the comparative analysis of fluorescence values,we determined the parameters for optimal fluorescence expression,and established the BiFC system with the porA promoter and the plasmid complementation of Venus fluorescent protein.The system was used to explore the interactions of flagellum-expressed proteins of C.jejuni.The results showed that it was known that the fluorescence values of BiFC-flhF-flhG vector,in which flhF interacted with flhG,were significantly higher than those of the negative control vector for in vivo expression of the bacterium,whereas BiFC-flhF-fliM and BiFC-flhF-fliG did not differ from the negative control,which indicated that the establishment of the BiFC system was successful,suggesting that the interactions of flhF with fliG and fliM did not have mutualistic relationship.In conclusion,the BiFC system for C.jejuni was established in this study,which provides an efficient and convenient method for the protein interactions study of this bacterium.