[1]肖大平,郭鹰,张卫军,等.肠出血性大肠埃希菌O157∶H7 tccP基因敲除菌株的构建[J].第三军医大学学报,2009,31(10):875-878.
 XIAO Da-ping,GUO Ying,ZHANG Wei-jun,et al.Mutant construction of enterohaemorrhagic Escherichia coli O157∶H7 with tccP gene knockout[J].J Third Mil Med Univ,2009,31(10):875-878.
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
31卷
期数:
2009年第10期
页码:
875-878
栏目:
论著
出版日期:
2009-05-30

文章信息/Info

Title:
Mutant construction of enterohaemorrhagic Escherichia coli O157∶H7 with tccP gene knockout
作者:
肖大平郭鹰张卫军顾江王海光朱凤才毛旭虎
第三军医大学医学检验系临床微生物及免疫学教研室;江苏省疾病预防控制中心急传科
Author(s):
XIAO Da-ping GUO Ying ZHANG Wei-jun GU Jiang WANG Hai-guang ZHU Feng-cai MAO Xu-hu
Department of Clinical Microbiology and Immunology, College of Medical Laboratory Science, Third Military Medical University, Chongqing 400038; Center for Disease Prevention and Control of Jiangsu Province, Nanjing 210009, China
关键词:
肠出血性大肠埃希菌O157∶H7tccP同源重组基因敲除
Keywords:
EHEC tccP homologous recombination gene knockout
分类号:
Q78;Q933;Q939.121
文献标志码:
A
摘要:
目的   构建肠出血性大肠埃希菌O157∶H7介导和宿主细胞的粘附与擦拭(A/E)损伤相关基因tccP敲除菌株。   方法   根据GenBank公布的肠出血性大肠埃希菌O157∶H7基因序列,设计PCR引物,分别扩增克隆tccP基因两端外侧的DNA片段,并与氯霉素耐药基因Cam连接后亚克隆于pBluescriptSKⅡ质粒,通过同源重组替换野生型tccP基因,采用PCR、RT-PCR 以及Western blot分别从DNA、RNA及蛋白质水平鉴定tccP基因是否被敲出。   结果   在DNA、RNA及蛋白质3个水平均证实Cam片段已成功替换tccP基因而整合进入O157∶H7基因组中。   结论   成功构建肠出血性大肠埃希菌O157∶H7 tccP基因敲除菌株。
Abstract:
Objective   To construct an enterohaemorrhagic Escherichia coli (EHEC) strain with tccP gene knockout based on O157∶H7 strain, which gene mediates attaching and effacing (A/E) lesions.    Methods   According to EHEC O157∶H7 gene sequence in the GenBank, PCR primers were designed and then the DNA fragments outside tccP were amplified. The PCR products was linked to chloramphenicol-resistant gene Cam and then subcloned into plasmid pBluescriptSKⅡ(-). After digested by double enzymes, the homologous recombination DNA fragments was used to replace tccP wild-type gene. PCR, RT-PCR, as well as Western blotting were used to identify tccP gene knockout from the DNA, RNA and protein levels respectively.    Results   DNA, RNA and protein levels confirmed that the Cam fragment had been successfully replaced tccP gene and integrated into the O157∶H7 genome.    Conclusion   EHEC O157∶H7 tccP knockout strain is successfully constructed, which is helpful in the research on the pathogenic mechanism of the bacteria.

相似文献/References:

[1]余抒,顾江,曾浩,等.肠出血性大肠埃希菌O157∶H7黏附HeLa细胞模型的建立[J].第三军医大学学报,2007,29(20):1933.
 YU Shu,GU Jiang,ZENG Hao,et al.Establishment of an enterohemorrhagic Escherichia coli O157∶H7 attached HeLa cell model[J].J Third Mil Med Univ,2007,29(10):1933.

更新日期/Last Update: 2009-05-08