嵌合鞭毛蛋白在大肠杆菌BL21(DE3) 和沙门菌SL5928中的表达与组装分析

Expression and assembly of chimeric flagellins in Escherichia coli BL21(DE3) and Salmonella SL5928

摘要:

鞭毛蛋白根据能否组装可以表达为单体或多聚体。目前，关于它们之间的区别少有报道。文中，将重组质粒pET-fliC/M2e2分别导入大肠杆菌BL21(DE3) 和沙门菌SL5928中来表达嵌合鞭毛蛋白mfliC/M和pfliC/M，然后分析它们的组装特性。SDS-PAGE结果表明，两重组菌成功表达了嵌合鞭毛蛋白。透射电镜观察显示，在重组大肠杆菌表面未见鞭毛，而重组沙门菌表面呈现鞭毛。将嵌合蛋白纯化后，圆二色谱 (Circular dichroism，CD) 分析表明，两者的CD信号明显不同，pfliC/M的CD信号与野生型鞭毛蛋白一致，而mfliC/M基本上没有CD信号出现。动态光散射分析表明，mfliC/M的聚集程度明显低于pfliC/M。将嵌合蛋白转染小鼠腹腔细胞3 h后，两者均能诱导产生IL-1β，但mfliC/M组高于pfliC/M组。以上结果对于鞭毛蛋白表达形式的选择具有重要的参考价值。

Abstract:

Flagellin can be expressed in monomeric or polymeric form based on assembly. The difference of these two forms of flagellin is less studied. In this experiment, recombinant plasmid pET-fliC/M2e2 was transferred into Escherichia coli BL21(DE3) and Salmonella SL5928 to express chimeric flagellin, mfliC/M and pfliC/M, respectively, and then their assembly characteristics were analyzed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results indicated that the two recombinant bacteria could successfully express chimeric flagellin. The transmission electronic microscope observation showed that no flagella were found on the surface of recombinant E. coli, whereas it was found for recombinant Salmonella. After purification, distinct circular dichroism spectra between them were found and pfliC/M showed the similar structure as wild-type flagellin, but not for mfliC/M. The dynamic light scattering assay also indicated that the polymerization of mfliC/M was much lower than that for pfliC/M. Three hours after transfection into mouse peritoneal macrophages, both could induce interleukin 1β secretion, but mfliC/M is stronger than pfliC/M. These data will be helpful for the selection of expression form of flagellin.