肠出血型大肠埃希菌O157:H7 espF基因缺失株和回补株的构建

Construction of enterohemorrhagic Escherichia coli O157:H7 strains with espF gene deletion and complementation

目的利用Red同源重组系统敲除肠出血型大肠埃希菌的esp F基因及其核苷酸片段;建立以p BAD 33质粒为载体的esp F基因回补实验平台。方法设计1对同源臂引物扩增卡那霉素抗性打靶片段,将抗性打靶片段电转入含有PKD46质粒的EDL933w,在PKD 46介导的重组系统帮助下,打靶片段和菌体esp F基因发生同源重组,PCR和测序验证;PCR扩增esp F及其核苷酸片段的整个ORF区序列,将其克隆入p BAD33质粒,分别将重组质粒转入相应的缺失株,以构建出相应的回补突变株。采用RT-PCR的方法验证在相应的回补突变株中esp F及其核苷酸片段是否转录。结果构建了esp F基因及其核苷酸片段缺失的EHEC O157:H7 EDL933w突变菌株和相应的回补株,且esp F基因及其核苷酸片段在回补株中均发生转录。结论本研究运用Red重组系统构建了肠出血型大肠埃希菌O157:H7 esp F基因缺失株,并建立以p BAD33质粒为载体的EHEC O157:H7基因回补方法,为进一步研究肠出血型大肠埃希菌中esp F基因的调控机制奠定了基础。

Objective To construct enterohemorrhagic Escherichia coli（EHEC） O157：H7 strains with delection esp F gene and its nucleotide fragment and with esp F gene complementation. Methods A pair of homologous arm primers was designed to amplify the gene fragment of kanamycin resistance, which was transformed into EHEC O157：H7 EDL933 w strain via the PKD46 plasmid by electroporation. The replacement of the esp F gene by kanamycin resistance gene through the PKD46-mediated red recombination system was confirmed by PCR and sequencing. The entire coding region of esp F along with its nucleotide fragment was amplified by PCR and cloned into p BAD33 plasmid, which was transformed into a mutant strain to construct the strain with esp F complementation. RT-PCR was used to verify the transcription of esp F and its nucleotide fragment in the complemented mutant strain. Results and Conclusion We established EHEC O157：H7 EDL933 w strains with esp F gene deletion and with esp F gene complementation. Both esp F and its nucleotide fragment were transcribed in the complemented mutant strain. The two strains provide a basis for further study of the regulatory mechanism of esp F.