缺失HPI毒力岛的EAggEC O42突变菌株的构建及鉴定

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (11): 1352-1356.

缺失HPI毒力岛的EAggEC O42突变菌株的构建及鉴定

胡静¹, 俞守义¹, 阚飙², 刘志华³

1. 南方医科大学流行病学教研室, 广东, 广州, 510515;
2. 中国疾病预防控制中心传染病研究所, 北京, 102206;
3. 南方医科大学南方医院感染内科, 广东, 广州, 510515

出版日期:2005-11-20 发布日期:2005-11-20
基金资助:
收稿日期:2004-9-28。
基金项目:国家重点基础研究发展规划项目资助(G1999054101)
作者简介:胡静(1972-),女,讲师,博士,E-mail:jing-hu@163.net

Construction and characterization of mutant Enteroaggregative E. coli O42 strain with high-pathogenicity island deletion

HU Jing¹, YU Shou-yi¹, KAN Biao², LIU Zhi-hua³

1. 南方医科大学流行病学教研室, 广东, 广州, 510515;
2. 中国疾病预防控制中心传染病研究所, 北京, 102206;
3. 南方医科大学南方医院感染内科, 广东, 广州, 510515

Online:2005-11-20 Published:2005-11-20

摘要/Abstract

摘要： 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pCO85。以EAggECO42为出发菌株,构建缺失irp8～irp5约24kb的HPI毒力岛功能核心区区域的全岛缺失株EAG85。结果通过接合转移和同源重组,利用蔗糖抗性筛选,pCO85上的同源序列有效的置换了EAggECO42的irp8和irp5基因。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毒力岛基因的EAggEC菌株。结论成功地构建了缺失HPI毒力岛的EAggECO42突变菌株,为进一步阐明HPI毒力岛在EAggEC菌株中的功能建立了重要的物质基础。

Abstract: 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.

中图分类号:

R-33

胡静¹, 俞守义¹, 阚飙², 刘志华³. 缺失HPI毒力岛的EAggEC O42突变菌株的构建及鉴定[J]. 南方医科大学学报, 2005, 25(11): 1352-1356.

HU Jing¹, YU Shou-yi¹, KAN Biao², LIU Zhi-hua³. Construction and characterization of mutant Enteroaggregative E. coli O42 strain with high-pathogenicity island deletion[J]. Journal of Southern Medical University, 2005, 25(11): 1352-1356.

参考文献

[1] Nataro JP. Kaper JB. Diarrheagenic Escherichia Coli[J]. J Clin Microbiol, 1998, 11(1): 142-201.
[2] Schubert S, Rakin A, Karch H, et al. Prevalence of the "highpathogenicity island" of Yersinia species among Escherichia coli strains that are pathogenic to humans[J]. Infect Immun, 1998, 66(2): 480-5.
[3] Gophna U, Oelschaeger TA, Hacker J, et al. Yersinia HPI in septicemic Escherichia coli strains isolates from diverse hosts[J].FEMS Microbiol Lett, 2001,196(1): 57-60.
[4] Koczura R, Kaznowski A. Occurrence of the Yersinia highpathogenicity island and iron uptake systems in clinical isolates of Klebsiella pneumoniae[J]. Microb Pathog, 2003, 35(5): 197-202.
[5] Oelschlaeger TA, D. Zhang, S. Schubert, et al. The high-pathogenicity island is absent in human pathogens of Salmonella enterica subspecies I but present in isolates of subspecies Ⅲ and Ⅵ[J]. J Bacteriol, 2003, 185(3): 1107-11.
[6] Camiel E, Guilvout I, Prentice M. Characterization of a large chromosomal "high-pathogenicity island" in biotype lB Yersinia enterocolitica[J]. J Bacteriol, 1996, 178 (23): 6743-51.
[7] Pelludat C, Rakin A, Jacobi CA, et al. The rersiniabactin biosynthetic gene cluster of Yersinia: organization and siderophore-dependent regulation[J]. J Bacteriol, 1998, 180(3): 538-46.
[8] Rakin A,Noelting C, Schubert S, et al. Common and specific characteristics of the high-pathogenicity island of Yersinia enterocolitica[J]. Infect Immun, 1999, 67(10): 5265-74.
[9] Rakin A, Saken E, Harmson D, et al. The pesticin receptor of Yersinia enterocolitica: a novel virulence factor with dual function[J]. Mol Microbiol, 1994, 13(2): 253-63.
[10] 金冬雁,黎孟枫(编译).分子克隆实验指南[M].第2版.北京:科学出版社,1993.
[11] 于永茂,刘延清,祁国明,等.霍乱A-B+减毒活疫苗的构建[J].中华流行病学杂志(Chin J Epidemiol),1993,14(Sup15):136-41.

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