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Westernblot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∶H7tccP基因敲除菌株。
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 pBluescriptSK Ⅱ （ - ）. After digested by double enzymes, the homologous recom- bination 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 constrncted, which is helpful in the research on the pathogenic mechanism of the bacteria.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2009年第10期875-878,共4页
Journal of Third Military Medical University
基金
国家高技术研究发展计划(863计划)(2007AA02Z409)
国家自然科学基金(30670107)
重庆市自然科学基金(2006BB5115)~~
