肠出血型大肠埃希菌O157:H7 ppk基因缺失株的构建及其生物学特性分析  

Construction and characterization of enterohemorrhagic Escherichia coli O157:H7 ppk-deleted strain

作　　者：韩朋[1] 孙琦[1] 赵素慧[1] 张其威[1] 万成松[1] 

机构地区：[1]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

出　　处：《南方医科大学学报》2014年第6期904-908,共5页Journal of Southern Medical University

基　　金：国家自然科学基金(81371765);广东省自然科学基金(S2013010014393)~~

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 O157: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 O157: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 O157: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 O157:H7 EDL933w突变菌株(EHEC O157:H7 EDL933wΔppk),初步探索了EHEC O157: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。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＆#39; and 3＆#39; 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, OD60 value and Giemsa staining. Results and Conclusion We established a ppk-deleted EHEC O157：H7 EDL933w strain with kanamycin resistance and compared the biological characteristics of the wild-type and mutant strains, which may facilitate further study of the regulatory mechanism of ppk gene.

关 键 词：EHEC O157 H7 ppk基因 RED重组系统 基因突变 

分 类 号：R378.21[医药卫生—病原生物学] R346[医药卫生—基础医学]