根据GenBank中毒素基因vt1、vt2序列设计合成4 对引物,以大肠杆菌O157 菌株DNA为模板,扩增vt1、vt2,从只含有vt2的菌株中诱导释放噬菌体,以噬菌体DNA为模板进行PCR扩增,获得vt2、vt2 A、vt2 B 3条特异性DNA带;将vt2 A、vt2 B扩增产物纯化后,分别插入pMD18 T载体,测序结果与相应序列比较,vt2 A和vt2 B亚单位的基因序列与GenBank中编码VT2 毒素的A、B亚单位的核苷酸序列(X07865,NC_002655, BA000007,AF291819)的同源性分别为98%~99%、96%~100%,确定vt2 位于噬菌体,并为进一步研究大肠杆菌O157 中VT噬菌体的毒力转导、VT2毒素的表达和应用奠定基础。

According to the nucleotide sequences of the vt1 and vt2 toxin genes in GenBank, four pair of primers were designed and synthesized. vt1 and vt2 were detected in E.coli O157:H7 strains. Bacteriophage was induced and released from the strain only carrying vt2. Phage DNA was extracted and used as PCR template for the detection of the vt2, vt2-A and vt2-B genes. The purified vt2-A and vt2-B fragments were inserted into pMD18-T vector. Sequencing proved that two sequences were genes of subunit A and B of vt2. The homologies of two sequences compared to their corresponding sequences coding VT2 toxin in GenBank (accession numbers X07865, NC_002655, BA000007, AF291819) were 98%~99%, 96%~100%,respectively. Our results revealed that vt2 gene was encoded by bacteriophage, which provides a foundation for further research on horizontal transmission of VT phage carrying virulent gene.