肠出血性大肠埃希菌O157噬菌体的生物学特性

目的：分离并鉴定肠出血性大肠埃希菌O157噬菌体，对其生物学特性进行研究，为开发抗肠出血性大肠埃希菌O157的生物制剂提供实验依据。方法：以肠出血性大肠埃希菌O157为宿主菌，采用噬斑法从环境污水中分离出针对肠出血性大肠埃希菌O157的噬菌体。噬菌体经超滤浓缩后，电镜观察其大小和形态；将宿主菌和噬菌体以不同的比例混合，测定噬菌体的最佳感染复数并观察其一步生长曲线；利用SDS-PAGE分析噬菌体的结构蛋白和非结构蛋白；提取噬菌体基因组，进行酶切电泳分析。结果：电镜显示噬菌体的头部呈球形、直径40 nm，噬菌体的尾部长约80 nm。噬菌体的最佳感染复数为10^-2,即当宿主菌和噬菌体之间的比例为1∶100时裂解效率最高。一步生长曲线示噬菌体在裂解宿主菌时，潜伏期约为10 min，爆发期约为30 min，裂解量为130。SDS-PAGE凝胶电泳呈现13种蛋白，相
对分子质量为16 000～22 000。 琼脂糖凝胶电泳显示噬菌体基因组大小约23 000 bp。结论：成功分离并鉴定一株针对肠出血性大肠埃希菌O157的噬菌体，是一种裂解性强、特异性高的毒性噬菌体，肠出血性大肠埃希菌O157噬菌体属于有尾病毒目，管尾噬菌体科。

Biological characteristics of enterohemorrhagic E.coli O157-specific bacteriophages isolated from raw sewage

Objective To isolate enterohemorrhagic E.coli O157 phages from raw sewage and to investigate the biological characteristics of isolated phages and to provide experimental basis for the development of a biologic agent against enterohemorrhagic E.coli O157.Methods Bacteriophages were isolated from raw sewage and were identified by plaque methods using enterohemorrhagic E.coli O157 as host cells.After purification of phages with ultrafitration,the size and morphology of phages were examined by electron microscope,and the multiplicity of infection and growth curve of phages were carried out by mean of host bacteria and phages mixture in different portions.The structural and nonstructural proteins of phages were analyzed by SDS-PAGE electrophoresis.The extracted genomes of phages were digested with restriction enzymes.Results The results of electron microscope showed that the head of phages exhibited a spherical form,40 nm in diameter and these phages had a tail,about 80 nm in length.The multiplicity of infection of phages was 10-2,that was,when the ratio of host bacteria to phages being 1∶100,the most efficient lysis.The growth curve of phages revealed that the incubation was 10 min,the outbreak of the time was 30 min and the lysis amount was 130.SDS-PAGE analysis showed that 13 kinds of proteins of phages were found and their relative molecular mass were 16 000-20 000.Electrophoresis of DNA showed that the size of the genome of phages was 23 000 bp.Conclusion A strain of bacteriophages against enterohemorrhagic E.coli O157 is isolated and characterized,and it is lytic and highly specific to E.coli O157.According to the biologic features,enterohemorrhagic E.coli O157 phages are classified into Caudovirales,Siphoviridae.