出血性大肠杆菌O157菌壳的制备研究

目的利用温度控制噬菌体PhiX174裂解基因E的表达,制备出血性大肠杆菌O157∶H7菌壳,鉴定其裂解效率并观察其形态。方法利用PCR技术扩增得到噬菌体PhiX174裂解基因E并克隆到质粒pBV220中,将重组质粒导入出血性大肠杆菌O157∶H7。重组菌株O157∶H7(pBV220::E)培养温度从28℃突升至42℃,每20min检测菌液OD600值,绘制重组菌株生长曲线。体外菌落计数计算裂解效率,并通过电镜观察菌壳结构。结果获得了PhiX174裂解基因E全长基因及重组质粒,构建了大肠杆菌的重组菌株。重组菌菌液OD600值在温度突升至42℃后40min后开始显著下降,80min后OD600值趋于平稳。细菌的裂解效率为98.4%。电子显微镜观察显示,经诱导后,O157∶H7细菌内容物可以通过细菌表面的孔道流出,从而形成菌壳。结论本研究成功制备了大肠杆菌O157∶H7菌壳,为将来进一步研究O157菌壳的特性奠定了基础。

Generation and characterization of enterohemorrhagic Escherichia coli O157 ghosts

Enterohemorrhagic Escherichia coli（EHEC）O157∶H7 is an important food borne pathogen capable of causing non-bloody or bloody diarrhea,hemorrhagic colitis（HC）and potentially fatal hemolytic uremic syndrome（HUS）.Many efforts have been made by several research groups to develop vaccine.However there is not any vaccine could be used in clinic.Bacterial ghosts are nonliving bacterial cell envelopes devoid of cytoplasmic contents while maintaining their cellular morphology and native surface antigenic structures.Therefore,bacterial ghosts could be the excellent vaccine candidates.In the present study,lysis gene E was amplified by PCR from phage PhiX174 and inserted into pBV220 vector downstream of the λPL/PR-cI857 regulatory system to form the temperature-sensitive bacteriolysis plasmid（pBV220：：E）.Plasmid pBV220：：E was transformed into O157∶H7 and grown at 28℃ until mid log-phase,followed by incubation at 42℃ to induce the expression of gene E and generate O157∶H7 bacterial ghosts.The OD600 value of culture began to decline in 40 min after the temperature shift from 28℃ to 42℃.The lysis rate of culture was 98.4%.The O157∶H7 ghosts were shown to be intact cells under electron microscope,with contents released to extracellular region.In conclusion,the O157∶H7 ghosts were successfully generated with lysis gene E,which laid foundation for future development of bacterial ghost vaccine against EHEC O157∶H7 infection.