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Published on: November 12, 2012
[Construction and characterization of mutant Enteroaggregative E. coli O42 strain with high-pathogenicity island
Jing Hu1, Shou-yi Yu, Biao Kan
1Department of Epidemiology, Southern Medical University, Guangzhou 510515, China. jing-hu@163.net
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.
