缺失耶尔森菌HPI irp6基因EAggEC的构建
Constructing in-frame deletion irp6 gene mutant of enteroaggegative escherichia coli with yersinia HPI
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摘要:目的 构建小肠结肠炎耶尔森菌强毒力岛irp6重组自杀质粒, 并应用其构建EAggEC上该基因的缺失株。方法 根据已知小肠结肠炎耶尔森菌强毒力岛irp6基因序列, 设计PCR引物, 扩增并克隆了irp6结构基因, 在体外进行精确突变后, 插入氯霉素抗性基因。再将含氯霉素的irp6缺失基因亚克隆入自杀质粒pKNG101中, 构建成irp6基因重组自杀质粒pKH6, 并以肠粘附聚集性大肠杆菌EAggEC17-2为出发菌株, 通过体内同源重组, 置换其irp6基因。结果 经接合转移和同源重组, 利用蔗糖抗性筛选, pKH6上的同源序列有效的置换了EAggEC17-2的irp6基因。结论 以小肠结肠炎耶尔森菌为靶构建的irp6基因重组自杀质粒成功的缺失了EAggEC17-2的irp6基因, 获得了携带耶尔森菌强毒力岛的EAggEC17-2irp6基因缺失株EAG6, 为进一步研究其功能打下了基础。Abstract:Objective To construct the recombinant suicide vectors of irp6 gene in yersinia HPI and use it to construct in-frame deleted mutant of EAggEC.Methods According to the sequence of irp6 gene of yersinia enterocolitica, the PCR primers were designed to amplify irp6 gene, then the PCR fragment was cloned into pMD18T-vector.The recombinant plasmid containing irp6 gene was mutated by precise deletion of middle fragment of irp6 gene in vitro. Then chloramphenicol gene was inserted into it.The fragment containing in-frame deleted irp6 gene which was inserted by chloramphencol gene was subcloned into suicide vector pKNG101, named pKH6.Moreover, EAggEC 17-2 was used to construct irp6 gene mutant by homologous recombination.Results Irp6 gene of EAggEC 17-2 was exchanged by pKH6 by conjunction mobilization with the selection of sucrose.Conclusion pKH6 which was constructed from the sequence of yersinia enterocolitica resulted in the construction of the in-frame deletion irp6 gene mutant EAG6 of EAggEC 17-2 successfully. The research on irp6 gene function would be carried out with EAG6.
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