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目的:用Red系统快速敲除肠出血性大肠杆菌O157∶H7中的分子伴侣基因,构建EHEC O157∶H7的突变体。方法:本研究首先合成一对引物(每一条的5′端与ecs4553或者ecs4563基因同源,3′端与卡那霉素抗性基因同源),用pKD4为模板扩增出带有卡那霉素抗性基因的PCR产物,然后电击转化至大肠杆菌中,在λRed重组系统的帮助下,通过卡那霉素抗性基因两侧的ecs4553或者ecs4563基因序列在体内与ecs4553或者ecs4563基因发生同源重组,置换掉基因组中的ecs4553或者ecs4563基因,最后利用卡那霉素抗性基因两端的FRT位点,通过FLP位点专一性重组将卡那霉素抗性基因剔除。结果:获得了ecs4553或者ecs4563基因被精确敲除且不带卡那霉素抗性基因的EHEC O157∶H7突变菌株。结论:为进一步研究EHEC O157∶H7的致病机制奠定了基础。
OBJECTIVE: To rapidly knock out the molecular chaperone gene in enterohemorrhagic Escherichia coli O157: H7 by Red system to construct a mutant of EHEC O157: H7. Methods: In this study, we first synthesized a pair of primers (the 5 'end of each is homologous to the ecs4553 or ecs4563 gene, and the 3' end is homologous to the kanamycin resistance gene). Using pKD4 as a template, The PCR products of the resistance genes were electroporated into Escherichia coli and transformed with the ecs4553 or ecs4563 gene in vivo by the ecs4553 or ecs4563 gene sequences flanking the kanamycin resistance gene with the help of the lambda Red recombination system Source recombination, replacing the ecs4553 or ecs4563 gene in the genome, and finally using the FRT site at both ends of the kanamycin resistance gene to eliminate the kanamycin resistance gene by FLP site-specific recombination. Results: EHEC O157: H7 mutant strains were obtained which were knocked out ecs4553 or ecs4563 gene without kanamycin resistance gene. Conclusion: This study laid the foundation for further study on the pathogenesis of EHEC O157: H7.