粘质沙雷菌裂解性噬菌体ФSM9-3Y的分离和鉴定

目的从医院污水中分离粘质沙雷菌噬菌体,并分析其生物学特性,为进一步研究针对耐药性粘质沙雷菌的噬菌体制剂提供依据。方法采用双层琼脂平板法分离纯化针对粘质沙雷菌的裂解性噬菌体,观察噬菌体对宿主菌的裂解特异性,通过负染法电镜观察噬菌体的形态结构,提取噬菌体核酸进行酶切电泳,测定噬菌体的最佳感染复数和一步生长曲线,SDS-PAGE电泳初步分析噬菌体的结构蛋白和非结构蛋白。结果从医院污水分离出7株可裂解粘质沙雷菌的噬菌体,对其中一株噬菌体(命名为ФSM9-3Y)的生物学特征进行了初步研究。电镜显示噬菌体呈蝌蚪状,头部为20面体立体对称、直径约70 nm;尾部长约50 nm。ФSM9-3Y的最佳感染复数为1。一步生长曲线表明;ФSM9-3Y的潜伏期约30 min,暴发时间70 min,暴发量为629 PFU/cell。凝胶电泳显示噬菌体基因组为双链DNA、大小约54 kb。SDS-PAGE呈现至少包括13种蛋白,相对分子质量范围在25～130 kD,其中主要蛋白的相对分子质量约为48 kD。结论此次分离的噬菌体ФSM9-3Y为裂解性噬菌体,根据形态和结构特征,粘质沙雷菌噬菌体ФSM9-3Y属于有尾病毒目,肌尾噬菌体科。

Isolation and characterization of a novel lytic phage ФSM9-3Y of Serratia marcescens

Objective To explore the biological characteristics of Serratia marcescens phages isolated from sewage for a further investigation to develop phage agents against drug-resistant Serratia marcescens.Methods Lytic phages of Serratia marcescens were isolated and identified by using a double-agar method,the specificity of phages to host cells was observed.Phage samples for observation by electron microscopy were carried out with negative-stain.The extracted genomes of lytic phages were analyzed with restriction enzymes on electrophoresis,the multiplicity of infection and one-step growth experiment of phages were evaluated.The protein patterns of phages were completed by SDS-PAGE.Results 7 lytic phages of Serratia marcescens were isolated from raw sewage,one of these,ФSM9-3Y,was further studied.Electron microscopy revealed ФSM9-3Y having an icosahedral head(70 nm) and a tail(50 nm in length).The multiplicity of infection(MOI) of ФSM9-3Y was 1,the one-step growth curve of ФSM9-3Y showed that the latent-period is 30 min,burst-period is 70 min,and the burst size is 629 PFU/cell.DNA electrophoresis indicated that the genome is double-stranded DNA estimated to be 54 kb in size.SDS-PAGE demonstrated a total of 13 protein bands with a relative molecular quality ranging from 25 to 130 kD.A major protein band,with a relative molecular quality of 48 kD,was observed.Conclusion The phage ФSM9-3Y was capable of efficient lysis of Serratia marcescens.According to its biological features,the lytic phage,ФSM9-3Y,belongs to Caudovirale order,Myoviridae family.