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目的:利用温度控制噬菌体PhiX174裂解基因E的表达,制备大肠杆菌细菌菌蜕,鉴定其裂解效率并观察其形态。方法:利用PCR技术扩增分离了噬菌体PhiX174裂解基因E并构建其表达质粒,并将质粒转化大肠杆菌DH5α构建工程菌。工程菌培养温度从28℃突升至42℃,每15 min检测菌液D600值,诱导后4 h收集菌体。体外菌落计数计算裂解效率,并通过电镜观察菌蜕结构。结果:获得273 bp的PhiX174裂解基因E全长基因及表达质粒。菌液D600在温度突升至42℃后30 min开始持续下降,2 h后基本维持不变。4 h后收集菌体测得裂解效率为99.99%。电镜观察菌蜕为具有完整外膜结构的细菌空壳,并且无细胞毒性。结论:本研究成功利用噬菌体PhiX174裂解基因E实现了大肠杆菌细菌菌蜕的制备,为进一步的疫苗及递送系统研究奠定了基础。
OBJECTIVE: To use the temperature-controlled phage PhiX174 to lyse the expression of gene E to prepare E. coli bacterial spores, and to identify its lysis efficiency and observe its morphology. Methods: PCR was used to amplify and isolate the phage PhiX174 lytic gene E and construct its expression plasmid. The plasmid was transformed into E. coli DH5α to construct engineering bacteria. Engineering bacteria culture temperature rose from 28 ℃ to 42 ℃, the D600 value was detected every 15 min, and the cells were collected 4 h after induction. The colony counting was used to calculate the lysis efficiency, and the bacterial slough structure was observed by electron microscope. Results: The full-length gene and expression plasmid of PhiX174 cleavage gene was obtained. Bacterium liquid D600 began to drop continuously at 30 min after the temperature rose to 42 ℃, and remained unchanged after 2 h. After 4 h collection of cells measured cleavage efficiency of 99.99%. Electron microscopy bacterial sloughs have a complete outer membrane structure of the bacterial shell, and no cytotoxicity. Conclusion: The study successfully used the bacteriophage PhiX174 to cleave the gene E to prepare E. coli bacterial spores, which laid the foundation for further vaccine and delivery system research.