肠出血性大肠杆菌O157∶H7前噬菌体CP-933Y缺失突变株的构建  

Construction of Prophage CP-933Y Deletion Mutant Strain of Enterhemorrhagic E.coli O157:H7

作　　者：张传朋[1,2] 刘雄[2] 李涛[2] 王慧[1,2] 

机构地区：[1]广西医科大学,广西南宁530021 [2]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071

出　　处：《生物技术通讯》2016年第6期804-807,共4页Letters in Biotechnology

基　　金：国家重点基础研究发展计划(2015CB554202);国家自然科学基金(81401643)

摘　　要：目的:利用Red重组系统敲除肠出血性大肠杆菌O157∶H7前噬菌体片段CP-933Y,进而构建CP-933Y缺失突变株。方法:以肠出血性大肠杆菌O157∶H7菌株为模板,加入酶切位点PCR扩增前噬菌体CP-933Y上、下游各600 bp的同源臂序列;酶切后分别连接到p UC19-kan质粒的卡那霉素(包含FRT位点)抗性基因两侧,构建中间是卡那霉素抗性基因标记含有目的基因上、下游同源序列的线性片段;导入含有p KD46质粒的O157∶H7菌株中,利用Red编码的同源重组酶使该片段与目的基因上、下游发生同源重组,卡那霉素抗性基因置换菌株中CP-933Y前噬菌体片段,最后导入p CP20质粒去除卡那霉素抗性标记基因。结果:经PCR及测序验证,O157∶H7菌株中前噬菌体片段CP-933Y被敲除,敲除株与野生株具有相似的生长曲线。结论:构建了大肠杆菌O157∶H7前噬菌体CP-933Y缺失株,为进一步研究前噬菌体CP-933Y的功能奠定了基础。Objective： To knockout the prophage CP-933 Y fragment in enterohemorrhagic Escherichia coli（EHEC）O157∶H7 with Red recombination system to construct CP-933 Y deletion mutant. Methods： The EHEC O157∶H7strain was used as template, and 600 bp up and down homology arms of the prophage CP-933 Y with added en-zyme sites were amplified by PCR respectively. After enzyme digestion, the two arms were inserted in p UC19-kanplasmid（including FRT sites） on each end of the kanamycin resistance gene respectively to construct the linearfragment, and then it was tranfered into EHEC EDL933 strain containing p KD46 plasmids. The kanamycin resis-tant gene replaced the target gene CP- 933 Y by Red system in EHEC strain, and it was removed by followingtransformed p CP20 plasmid. Sequencing and PCR were used to identified the mutant strain, and the growth curveswere compared between the wild strain and the gene deletion mutant. Results： The deletion mutant stain was con-firmed by sequencing and PCR, and its growth was similar with that of wild strain. Conclusion： EHEC O157∶H7prophage CP-933 Y deletion strain construction laid the foundation for further stud of prophage fragment CP-933 Yfunction.

关 键 词：肠出血性大肠杆菌 前噬菌体 CP-933Y RED重组系统 

分 类 号：Q784[生物学—分子生物学]