奇异变形杆菌噬菌体的分离鉴定及其生物学特性分析

Isolation,identification and biological characters of bacteriophages of Proteus mirabilis

目的分离鉴定奇异变形杆菌噬菌体,并分析其生物学特性。方法以奇异变形杆菌为宿主菌,采用双层琼脂噬斑法从居民区生活污水中分离针对奇异变形杆菌的噬菌体;噬菌体经纯化后,通过负染法电镜观察其形态及大小;将噬菌体和宿主菌以10∶1的比例混合共培养,测定噬菌体的一步生长曲线;提取噬菌体核酸并进行酶切分析。结果共分离出4株噬菌体,分别命名为PrM-01、PrM-02、PrM-03和PrM-04,在双层琼脂平板上可形成圆形、透明、边界清晰、直径约2～3 mm的噬斑,经反复纯化后,其滴度范围在(1.2～2.3)×1012PFU/ml;电镜观察显示,噬菌体的头部呈二十面体立体对称,直径约40 nm,尾部长约30 nm;PrM-04的一步生长曲线表明,噬菌体在裂解宿主菌时,潜伏期为15 min,暴发时间为25 min,裂解量为3.2×104;酶切分析显示,其基因组大小约为26 000 bp,且具有EcoRⅠ、HindⅢ和BamHⅠ多个酶切位点。结论成功分离出4株针对奇异变形杆菌的噬菌体,其表现出特异性高、裂解性强的毒性噬菌体特征。

Objective To isolate and identify the bacteriophages of Proteus mirabilis,and analyze its biological characters.Methods Bacteriophages specific to P.mirabilis were isolated from the raw sewage by double-agar plaque method using P.mirabilis as host cells,then purified and observed for morphology and size by electron microscopy using negative staining.The bacteriophages and host cells were co-cultured at a ratio of 10 ∶ 1,based on which the one-step growth curve of bacteriophages was plotted,and the bacteriophage DNA was extracted and digested with restriction endonucleases.Results Four bacteriophage strains named as PrM-01,PrM-02,PrM-03 and PrM-04 were isolated,which formed round and transparent plaques with clear borders on double-agar plate,at diameters of 2 ～ 3 mm.The titers of bacteriophages after repeat purification were（1.2 ～ 2.3） × 1012 PFU / ml.Electron microscopy showed that the head of bacteriophages exhibited an icosahedra symmetry,at a diameter of about 40 nm,while the tail was at a length of about 30 nm.The one-step growth curve of PrM-04 revealed incubation period,time of outbreak and amount of lysis of 15 min,25 min and 3.2 × 104 respectively.Restriction analysis showed that the genome of bacteriophages was at a length of about 26 000 bp,with restriction sites of EcoRⅠ,Hind Ⅲ and BamHⅠ.Conclusion Four bacteriophage strains specific to P.mirabilis were successfully isolated,which showed characters of virulent bacteriophages,such as high specificity and strong lytic ability.