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目的分离鉴定大肠埃希菌噬菌体并分析其裂解特性,为噬菌体疗法应用于大肠埃希菌感染提供实验依据。方法采用双层琼脂噬斑法从污水中分离噬菌体,通过透射电镜观察噬菌体的形态学特征,利用限制性酶切图谱初步分析噬菌体的基因组,测定噬菌体对宿主菌的最佳感染复数和一步生长曲线,分析噬菌体对宿主菌的裂解谱,观察噬菌体在不同的pH及温度下对宿主菌的裂解特性,SDS-PAGE分析噬菌体的主要和次要蛋白。结果通过噬斑法从污水中分离出1株能裂解大肠埃希菌的噬菌体,命名为ΦEc-SL25;电镜显示,噬菌体ΦEc-SL25的形态特征符合有尾病毒目、管尾病毒科噬菌体;ΦEc-SL25的最佳感染复数为0.01;一步生长曲线表明,噬菌体ΦEc-SL25的潜伏期为5 min,爆发期为10 min;ΦEc-SL25对26株大肠埃希菌的裂解率可达30.8%;在温度70℃20min时以及在pH 4~10的范围内,噬菌体ΦEc-SL25仍保持其裂解活性;蛋白电泳可观察到2条主要蛋白带和至少3条次要蛋白。结论噬菌体ΦEc-SL25是一种潜伏期短、裂解较性强的毒性噬菌体,可用于开发针对大肠埃希菌感染的生物制剂。
OBJECTIVE: To isolate and identify Escherichia coli phage and analyze its lytic characteristics to provide experimental evidence for phage therapy in Escherichia coli infection. Methods Bacteriophages were isolated from wastewater by double-layer agar-plaque method. Morphological characteristics of the phages were observed by transmission electron microscopy. The phage genomes were analyzed by restriction enzyme digestion, and the optimal multiplicity of infection and one-step growth curve , The fragmentation pattern of the phage against the host bacteria was analyzed, the lytic characteristics of the phage against the host bacteria at different pH and temperature were observed, and the major and minor proteins of the phage were analyzed by SDS-PAGE. Results Phage was isolated from wastewater by plaque assay and named as ΦEc-SL25. The morphology of phage ΦEc-SL25 was found to be consistent with that of the phage mycoides, -SL25 was 0.01. One-step growth curve showed that phage ΦEc-SL25 had an incubation period of 5 min and an outbreak period of 10 min. The rate of ΦEc-SL25 against 26 strains of Escherichia coli was up to 30.8% Phage ΦEc-SL25 still maintained its lytic activity at 70 ℃ for 20 min and in the range of pH 4 ~ 10. Two major protein bands and at least three minor proteins were observed by electrophoresis. Conclusion The bacteriophage ΦEc-SL25 is a kind of toxic phage with short incubation period and relatively strong lysis. It can be used to develop biological agents against Escherichia coli.