大肠杆菌O157:H7中编码vt2基因的噬菌体的分离与鉴定

Isolation and Identification of Bacteriophage Verotoxin 2 Gene from E. coli O157:H7

根据GenBank中VT1、VT2毒素的基因序列设计合成2对引物,以大肠杆菌O157:H7菌株DNA为模板,扩 增vt1、vt2。诱导只扩增出vt2的菌株释放噬菌体,利用多种指示菌经双层琼脂平板法来分离纯化VT2噬菌体,观 察噬菌斑的特征,提纯病毒粒子进行电镜观察,并对噬菌体中vt2基因检测、克隆和序列分析。结果显示VT2噬菌 体感染MC1061在双层琼脂平板上形成的噬菌斑小而混浊,多呈磨玻璃样;而首次感染大肠杆菌CC118(λpir),此 后用MC1061分离的噬菌体,再以MC1061为指示菌,在双层琼脂平板上形成小而清晰透明的噬菌斑。电镜下噬 菌体头部呈六边形外廓,尾部细长无尾鞘结构。以噬菌体DNA为模板进行PCR扩增,检测到vt2特异性DNA 带,克隆的vt2基因序列与GenBank中编码VT2毒素的核苷酸序列(X07865,NC_002655,BA000007,AF291819) 的同源性分别达到99％,确定编码VT2毒素的基因位于噬菌体上,并获得VT2噬菌体(?)HY。

According to the nucleotide sequences of the VT1 and VT2 toxin genes in GenBank, two pairs of primers were designed, synthesized and used to amplify vtl and vt2 in Escherichia coli O157： H7. The strain carrying only the vt2 gene was inducted by mitomycin C and the bacteriophage carrying vt2 （VT2 phage） was released. The induced phage was isolated and purified by the two-layer agar assay. The plaques were small and cloudy from the host strain MC1061. The phage plaques were clear when first replicated in CC118（λpir） then MC1061. Electron microscopy showed that purified phage had a hexagonal head,a thin and long tail and no caudal sheath. The vt2 gene from the purified phage DNA was cloned and sequenced. It had a 99 % identity with the nucleotide sequence the gene encoding VT2 toxin in GenBank （accession numbers X07865, NC_ 002655, BA000007, AF291819）. The results confirmed that vt2 gene was encoded by bacteriophage genome. The VT2 phageφHY was obtained and identified.