Isolation and Lysogenic Transfection of Verocytotoxigenic Bacteriophage in E.coli

YAN Ya-xian; LU Cheng-ping

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August 2004

YAN Ya-xian and LU Cheng-ping. Isolation and Lysogenic Transfection of Verocytotoxigenic Bacteriophage in E.coli[J]. Virologica Sinica, 2004, 19(4): 389-393.

Citation: YAN Ya-xian, LU Cheng-ping. Isolation and Lysogenic Transfection of Verocytotoxigenic Bacteriophage in E.coli .VIROLOGICA SINICA, 2004, 19(4) : 389-393.

大肠杆菌VT2噬菌体的分离与溶源转染*

严亚贤 ^1,, ,
陆承平 ²

1.
1. 上海交通大学农业与生物学院动物科学系，上海 201101
2.
南京农业大学农业部动物疫病诊断与免疫重点开放实验室，南京 210095

通讯作者： 严亚贤, ;

摘要

本试验利用指示菌MC1061，经双层琼脂法纯化和PCR扩增vt2基因，分别从大肠杆菌O157菌株、牛粪、鸡粪和污水中分离获得5株含vt2基因的噬菌体。这些噬菌斑透明，直径为0.5-2 mm，对指示菌的感染效价均在109 PFU/mL以上，抵抗氯仿和56℃30min的作用。将噬菌体分离株SHφW1感染MC1061后，经PCR鉴定获得一株溶源菌株(MC1061/SHφW1)。溶源株的LB培养滤液对Vero细胞产生了显著的病变效应，而MC1061在同等条件下培养的滤液无细胞病变，表明VT2噬菌体通过溶源将vt2毒力基因水平转移，证实了VT2噬菌体的转染与细菌毒力相关。
- 噬菌体
- , vt2基因
- , 转染
- , 细胞病变
- , 毒力转移

Isolation and Lysogenic Transfection of Verocytotoxigenic Bacteriophage in E.coli

YAN Ya-xian ^1,, ,
LU Cheng-ping ²

1.
1. College of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 201101,China
2.
Key Laboratory of Animal Disease Diagnostic & Immunology, Ministry of Agriculture, Nanjing Agricultural University, Nanjiang 210095,China

Corresponding author: YAN Ya-xian,

Abstract

5 strains of verocytotoxigenic (VT) bacteriophage were isolated from E.coli O157 strains, cow feces, chicken feces, sewage and purified by two-layer agar assay. Gene vt2 was amplified by PCR in these phage isolates. The plaques of these phages were 0.5-2mm in diameter and the titre of phages with lucidity plaques against host strain MC1061 was more than 109 PFU/mL. A lysogen with vt2 gene was obtained after transfecting MC1061 by VT2 phage SHФW1 isolated from sewage. The cytopathic effect on Vero cells was produced after inoculating the filtrate from the above lysogen, while no cytopathic effect was appeared inoculating the filtrate from MC1061 cultured in the same condition. All these showed that VT2 phages were common in environment and VT2 virulent factor could transfer horizontally through the lysogenic infection by VT2 phage. VT2 phage is related to the virulence of bacteria.
- Bacteriophage
- , vt2 gene
- , Transfection
- , Cytopathic effect
- , Virulence transfer

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通讯作者: 陈斌, bchen63@163.com

1.
沈阳化工大学材料科学与工程学院沈阳 110142

Isolation and Lysogenic Transfection of Verocytotoxigenic Bacteriophage in E.coli

YAN Ya-xian ^1,, ,
LU Cheng-ping ²

Corresponding author: YAN Ya-xian,

1. 1. College of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 201101,China
2. Key Laboratory of Animal Disease Diagnostic & Immunology, Ministry of Agriculture, Nanjing Agricultural University, Nanjiang 210095,China

Abstract: 5 strains of verocytotoxigenic (VT) bacteriophage were isolated from E.coli O157 strains, cow feces, chicken feces, sewage and purified by two-layer agar assay. Gene vt2 was amplified by PCR in these phage isolates. The plaques of these phages were 0.5-2mm in diameter and the titre of phages with lucidity plaques against host strain MC1061 was more than 109 PFU/mL. A lysogen with vt2 gene was obtained after transfecting MC1061 by VT2 phage SHФW1 isolated from sewage. The cytopathic effect on Vero cells was produced after inoculating the filtrate from the above lysogen, while no cytopathic effect was appeared inoculating the filtrate from MC1061 cultured in the same condition. All these showed that VT2 phages were common in environment and VT2 virulent factor could transfer horizontally through the lysogenic infection by VT2 phage. VT2 phage is related to the virulence of bacteria.