【摘要】 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。更多还原

【Abstract】 Objective To isolate and identify bacteriophages against Escherichia coli,analyze their lytic activities and provide basis for researches on phage therapy for E.coli infection.Methods Double-layer agar method was employed to screen lytic phages from sewage;their morphological characteristics were observed by using transmission electron microscope.The phage genome digested with endonucleases was subjected to electrophoresis;the multiplicity of infection(MOI)and one-step growth curve of the phages were obtained.The host range of phage was determined.The lytic activities of phages against host cells were detected under a wide range of pH values and temperatures.SDS-PAGE was used to analyze the protein profiles of the phages.Results A highly virulent bacteriopphage(designated asΦEc-SL25)against E.coli was isolated.Transmission electron microscope showed thatΦEc-SL25 may be classified as a member of Caudovirales order,Siphoviridae family.The MOI ofΦEc-SL25 was 0.01,and one-step growth curve revealed a short latency period of 5min and a rise period of 10 min.ΦEc-SL25 lysed 8of the 26(30.8%)clinical isolates of E.coli,and kept its lytic activity in a range of pH 3to 10 as well as at temperature of 70℃for 20 min.Analysis of protein profiles showed 2 major proteins and 3 minor proteins.Conclusion Given that phageΦEcSL25exhibited a short latency,a high specificity and strong virulence,it may be a candidate for development of therapeutic agent to control infectious diseases caused by E.coli.更多还原