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肠出血性大肠埃希菌O157:H7 tccP基因敲除菌株的构建

Da-ping XIAO,Ying GUO,Wei-jun ZHANG,Jiang GU,Hai-guang WANG,Feng-cai ZHU,Xu-hu MAO

Acta Academiae Medicinae Militaris Tertiae（2009）

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Abstract
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pBluescriptSK Ⅱ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.
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Key words
EHEC,tccP,homologous recombination,gene knockout
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