肠出血性大肠埃希菌O157:H7菌影生物学活性的初步研究

Initial research on bioactive of EHEC O157:H7 bacterial ghosts

目的制备肠出血性大肠埃希菌O157∶H7细菌菌影,在细胞及动物水平对其生物学活性进行初步评价。方法将包含噬菌体phiX174裂解基因E的重组质粒pLSE转化肠出血性大肠埃希菌O157∶H7 EDL933及大肠埃希菌DH5α,42℃诱导制备菌影。利用间接免疫荧光实验观察菌影主要抗原结构,并利用电镜观察菌影对Hep-2细胞的黏附。将菌影免疫小鼠后,采用间接ELISA法检测小鼠特异性抗体,并通过小鼠攻毒实验考察菌影的保护效果。结果成功构建了重组质粒pLSE,制备出O157∶H7及DH5α菌影。O157∶H7菌影保留了病原菌的主要外膜抗原,能紧密黏附细胞Hep-2但并不侵入细胞。O157∶H7菌影免疫雄性BALB/c小鼠诱导特异性IgG抗体的产生。攻击试验结果显示小鼠对致死剂量O157∶H7 EDL933强毒株的保护率达到80%。结论 O157∶H7菌影的成功制备及生物学活性的初步析为研制O157∶H7菌影疫苗奠定了基础。

Objective To analyze the initial bioactivity on cell and animal models with prepared EHEC O157 ： H7 bacterial ghosts. Methods The recombinant plasmid pLSE which contained lysis gene E of phiX174 was constructed and transformed into EHEC O157 ： H7 EDL933 and E. coli DH5a, respectively. The bacterial ghosts were prepared by the inducing of temperature-shift to 42℃. Fluorescence microscope was used to observe the main antigens on the surface of bacterial ghosts. The adherence between bacterial ghosts and Hep-2 cells was tested under scan- ning electron microscopy and transmission electron microscopy. Specific IgG of immunized mice was tested by ELISA, and the protection rate of bacterial ghosts against lethal dose challenge was performed on mice model. Results Plasmid pLSE was constructed successfully, and the O157 ： H7 and DHSa bacterial ghosts were prepared. The O157 ： H7 bacterial ghosts displayed complete antigens the same as pathogen O157 ： H7, and the O157 ： H7 bacterial ghosts could adhere to Hep-2 cells tightly, but not invade the cells. O157 ： H7 bacterial ghosts could induce specific IgG on BALB/c mice and provide 80% protection against the lethal dose challenge of O157 ： H7 EDL933. Conclusion The successful preparation and initial analysis of EHEC O157 ： H7 bacterial ghosts estab- lished basis for the further research of novel O157 ： H7 vaccine.