肠出血型大肠埃希菌o157h7ppk基因缺失株的-南方医科大学学报.pdfVIP

·904 · doi 10.3969/j.issn. 1673-4254.2014.06.30 J South Med Univ, 2014, 34(6): 904-908 基础研究 肠出肠出血型大肠埃希菌血型大肠埃希菌OO157157:H:H77 ppkppk 基因缺失株的构建及其生物基因缺失株的构建及其生物 学特性学特性分析分析 韩 朋，孙 琦，赵素慧，张其威，万成松 南方医科大学公共卫生与热带医学学院BSL-3 实验室，广东 广州 510515 摘要：目的利用Red 重组系统构建肠出血型大肠埃希菌（EHEC）O157:H7 ppk 基因缺失株,并研究其生物特性。方法 设计并合 成一对同源臂引物，每条引物5端与ppk 基因同源，3端与卡那霉素抗性基因同源，扩增卡那霉素抗性基因片段；制备含有pKD46 质粒的EHEC O 157:H7 EDL933w 感受态细菌，然后将该抗性基因片段电击转化入制备的感受态菌株中；利用pKD46 介导的 Red 重组系统，通过同源重组替换EHEC O 157:H7 EDL933w ppk 基因，PCR 和测序验证；通过革兰染色、测D600值、吉姆萨染色比 较EHEC O 157:H7 EDL933w 野生株和突变株的形态结构、生长能力和黏附性。结果 构建了ppk 基因缺失并含有卡那霉素抗性 的EHEC O 157:H7 EDL933w 突变菌株（EHEC O 157:H7 EDL933wΔppk ），初步探索了EHEC O 157:H7 EDL933w 野生株和突变 株的生物学特性。结论 本研究运用Red 重组系统构建了肠出血型大肠埃希菌O157:H7 ppk 基因缺失株，为进一步研究肠出血 型大肠埃希菌中ppk 基因的调控机制奠定了基础。 关键词：EHEC O 157:H7 ；ppk 基因；Red 重组系统；基因突变 Construction and characterization of enterohemorrhagic Escherichia coli O157:H7 ppk- deleted strain HAN Peng, SUN Qi, ZHAO Suhui, ZHANG Qiwei, WAN Chengsong Biosafety Level-3 Laboratory, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China Abstract: Objective To construct enterohemorrhagic Escherichia coli (EHEC) O157: H7 ppk gene deletion strains and study its biological characteristics. Methods The gene fragment of kanamycin resistance was amplified using a pair of homologous arm primers whose 5 and 3 ends were homologous with ppk gene and kanamycin resistance gene, respectively. EHEC O157: H7 EDL933w competent strains were prepared and transformed via electroporation with the amplification products. The ppk gene was replaced by kanamycin resistance gene using pKD46-mediated Red recombination system. The recombinant strain was confirmed by PCR and sequencing, and its morphology, growth ability and adhesion were assessed using Gram staining, OD600 value and Giemsa staining. R