一株奥奈达希瓦氏菌烈性噬菌体的分离、纯化及鉴定

Isolation, purification and identification of a Shewanella oneidensis MR-1 lytic phage

摘要:

【目的】从环境中分离获得希瓦氏菌烈性噬菌体，并对其性质进行研究。【方法】以4株希瓦氏菌为宿主菌，采用双层平板法从污水样品中分离得到奥奈达希瓦氏菌MR-1烈性噬菌体M1；观察噬菌斑特征；利用超速离心法浓缩M1颗粒，进一步用氯化铯密度梯度离心纯化；采用透射电子显微镜观察纯化的M1颗粒；提取M1核酸，通过核酸酶处理分析其核酸类型及结构；绘制一步生长曲线。【结果】噬菌体M1在双层平板上形成圆形的噬菌斑，清晰透明，边缘光滑，直径为2.3 mm?2.5 mm；经电镜观察，噬菌体M1头部呈二十面体，直径约为55 nm，尾长约为170 nm，尾部可收缩，属于肌尾噬菌体科(Myoviridae)；通过酶切分析表明噬菌体M1核酸为线形双链DNA；一步生长曲线显示该噬菌体感染后完成一个复制循环所需要的时间约为15?20 min。【结论】噬菌体M1属肌尾噬菌体科，研究结果为后续研究病毒在地球微生物成岩过程中所起的作用提供了实验材料。

Abstract:

[Objective] A lytic phage of Shewanella was isolated from the sewage and characterized. [Methods] Four strains of Shewanella bacteria were used as the hosts for the isolation of lytic phages from the sewage. A lytic phage against Shewanella oneidensis MR-1, which we named M1, was isolated by the double-layer agar culture method. The plaque morphology was examined. Phage particles of M1 were concentrated by ultracentrifugation and purified by CsCl gradient centrifugation. The morphology of M1 particles was examined by transmission electron microscopy. The genome of M1 was extracted and the nucleic acid was analyzed by enzyme digestion. One-step growth curve was measured. [Results] The plaques of M1 phage were round and transparent with a diameter of about 2.3 mm?2.5 mm. Transmission electron micrographs showed that M1 had an icosahedral head about 55 nm in diameter and a long contractile tail about 170 nm in length. Phage M1 belongs to the Myoviridae family. Its genome is made of linear double-stranded DNA. The infection cycle of M1 was about 15?20 min as indicated by the one-step growth curve. [Conclusion] Bacteriophage M1 belongs to the Myoviridae family. Our results provided materials for the investigation on the roles of bacteriophages in diagenetic process on the earth.