肠出血性大肠埃希菌O157H7tccP基因敲除菌株的构建.docVIP

文章编号：200812213 肠出血性大肠埃希菌O157:H7 tccP 基因敲除菌株的构建 肖大平1，,郭鹰1，张卫军1，顾江1，王海光1，朱凤才2，毛旭虎1﹡(1第三军医大学医学检验系临床微生物及免疫学教研室重庆4000382江苏省疾病预防控制中心南京210009) 目的:构建肠出血性大肠埃希菌O157:H7介导与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。 肠出血性大肠埃希菌(EHEC)O157:H7；tccP；同源重组；基因敲除 R378.2＋1，Q319＋2 A Mutant construction of Enterohaemorrhagic Escherichia Coli O157:H7 tccP gene Da-Ping XIAO1, Ying GUO1, Wei-Jun ZHANG1, Jiang GU1,Hai-Guang WANG1, Feng-Cai ZHU2, Xu-Hu MAO1(1Department of Clinical Microbiology and Immunology, College of Medical Laboratory Science, Third Military Medical University, Chongqing 400038, China; 2JiangSu Provincial Center for Disease Prevention and Control, Nanjing210009,China) Abstract Objective: To construct EHEC O157: H7 tccP gene knockout strain, which gene mediates attaching and effacing (A/E) lesions. Methods: According to GenBank EHEC O157: H7 gene sequence, design PCR primers were amplified gene cloning tccP at both ends of the DNA fragments outside, and chloramphenicol-resistant gene subcloning in the pBluescriptSKII(-) plasmid. 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-blot had been used to identify tccP gene knockout from the DNA, RNA and protein levels. Results: DNA, RNA and protein levels confirmed that the Cam fragment has been successfully replaced tccP gene and integrated into the O157: H7 genome. Conclusion: The successful construction of EHEC O157: H7 tccP knockout strain for research in TccP EHEC O157: H7 pathogenic mechanism is helpful. Key words EHEC; tccP; homologous recombination;gene knockout 肠出血性大肠埃希菌(EHEC)O157:H7为重要的人畜共患传染病原，其感染具有暴发流行、强烈的致病性与致死性和抗生素治疗加剧病情等特点，同时它还可作为细菌武器与生物恐怖战剂的原料，已成为全球性的公共卫生问题[1, 2]。但致病机理至今尚未完