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目的明确肺炎克雷伯菌噬菌体PhF168的生物学特性及其对小鼠重症败血症的疗效。方法电镜观察PhF168的形态,调查其噬菌谱并研究其一步生长曲线、温度和酸碱稳定性等生物学特性;提取PhF168的基因组,酶切鉴定其核酸类型,并初步估算其基因组的大小;建立小鼠重症败血症感染模型,观察PhF168治疗效果。结果 PhF168可在宿主菌F168上形成直径3~5mm、完全透明并带有较宽晕环的噬菌斑,为裂解性噬菌体。电镜下其颗粒呈有尾噬菌体目长尾噬菌体科的形态特征。PhF168的基因组为dsDNA,>40kb,含有EcoRⅠ和BamHⅠ限制性内切酶切位点。PhF168在宿主菌F168中的潜伏期为15min,裂解量为(64±5)PFU/细胞,在pH 4~9和<50℃下具良好稳定性。用PhF168治疗由其宿主菌F168感染所致的重症败血症小鼠,所有小鼠的生存期均在3d以上,7d存活率达40%。未治疗对照组小鼠1d内全部死亡。结论 PhF168杀菌作用高效,具有较好的热稳定性和酸碱稳定性,治疗宿主菌引起的小鼠重症败血症疗效显著,可作为治疗细菌感染的噬菌体候选株。
Objective To determine the biological characteristics of Klebsiella pneumoniae phage PhF168 and its therapeutic effect on severe sepsis in mice. Methods The morphology of PhF168 was observed by electron microscopy. The phage spectrum of PhF168 was investigated. The biological characteristics of PhF168, such as its growth curve, temperature and acid-base stability, were investigated. The genomic DNA of PhF168 was digested with restriction endonuclease to identify its genotype. To establish a mouse model of severe sepsis infection and observe the therapeutic effect of PhF168. As a result, PhF168 could form 3 to 5 mm in diameter on the host strain F168, which was completely transparent with a larger halo and was a lytic bacteriophage. Under the electron microscope, the particles showed a phage-like phage phage phage phage morphology. The genome of PhF168 is dsDNA,> 40 kb, containing EcoRI and BamHI restriction sites. The incubation period of PhF168 in host strain F168 was 15min, the amount of cleavage was (64 ± 5) PFU / cell, and was stable at pH 4-9 and <50 ℃. With PhF168 treatment of severe septicemia mice caused by its host F168 infection, all mice survived more than 3d, and the survival rate of 7d was 40%. The untreated control mice all died within 1d. Conclusion PhF168 has high bactericidal activity, good thermal stability and acid-base stability. It has significant therapeutic effect on severe sepsis caused by host bacteria, and can be used as a candidate bacteriophage for bacterial infection.