缺失HPI毒力岛的EAggEC O42突变菌株的构建及鉴定

目的构建并鉴定HPI毒力岛缺失的EAggEC突变株.方法运用基因重组方法，以irp8基因部分序列作为同源重组的一侧序列，irp5基因序列作为同源重组的另一侧序列，中间插入有卡那霉素（Km）抗性基因（kan）标记。以pCVD442作为载体，构建含有irp8部分序列和irp5基因的重组自杀质粒pC085。以EAggEC O42为出发菌株．构建缺失irp8-irp5约24kb的HPI毒力岛功能卡受心区区域的全岛缺失侏EAG85．结果通过接合转移和同源重组．利用蔗糖抗性筛选，pC085上的同源序列有效的置换rEAggEC O42的irp8和irp5基凶。PCR方法扩增irp3、ybtA、irp9等基因的结果显示，筛选出的EAG85确为缺失HPI毒力岛基闪的EAggEC菌株结论成功地构建了缺失HPI毒力岛的EAggEC O42突变菌株．为进一步阐明HPI毒力岛存EAggEC菌株中的功能建立了重要的物质基础。

Construction and characterization of mutant Enteroaggregative E. coli O42 strain with highpathogenicity island deletion

OBJECTIVE: To construct and characterize a mutant Enteroaggregative E. coli(EAggEC) O42 strain with in-frame deletion of high-pathogenicity island (HPI). METHODS: The kanamycin resistance gene (kan) was inserted between the sequences of irp8 and irp5 genes as the two homologous sequences for construction of the recombinant plasmid pCO85 by subcloning the recombined sequence into the suicide vector pCVD442. By homologous recombination and conjunction mobilization, EAO85 mutant with deletion of the core region of HPI about 24 kb spanning from irp8 to irp5 sequences was screened. RESULTS: Irp8 and irp5 genes of EAggEC O42 were exchanged by pCO85 by conjunction mobilization with the selection by sucrose. The EAO85 mutant was identified by their failure to yield PCR products with primers specific for the internal regions of HPI. CONCLUSION: We have successfully constructed a mutant of EAggEC O42 strain with HPI in-frame deletion, which may facilitate the exploration of the role of HPI in EAggEC strain.