一株多耐药撒坝猪致病性E. coli irp2基因缺失菌株的构建  

Construction Of irp2 Gene Deleted Bacteria Strain of a Multiple Drug Resistant Pathogenic E.Coli Isolated from Saba Piglets

作　　者：富国文 敖平星 单春兰 赵汝 刘超英 高洪 FU Guowen;AO Pingxing;SHAN Chunlan;ZHAO Ru;LIU Chaoying;GAO Hong(College of Veterinary Medicine,Yunnan Agricultural University,Kunming 650201,China)

机构地区：[1]云南农业大学动物医学院,云南昆明650201

出　　处：《家畜生态学报》2021年第4期23-28,共6页Journal of Domestic Animal Ecology

基　　金：国家自然科学基金资助项目(31660704,31960692)。

摘　　要：Red同源重组是大肠埃希菌基因编辑的一个重要工具,pKD46、pKD3/4和pCP20是该操作中常用的工程质粒,通常以氨苄青霉素、卡那霉素和氯霉素作为筛选标记。在对一株分离自腹泻撒坝仔猪的致病性大肠埃希菌进行irp2基因敲除时发现该菌对氨苄青霉素、氯霉素等多种抗生素不敏感,是一株多耐药菌株,给筛选带来了困难。为了解决这个问题,我们构建了携带博来霉素抗性的打靶片段和携带FLP酶的重组质粒pCas-FLP,成功实现了该菌株的irp2基因缺失,为进一步研究该基因致病机制提供了基础,同时也为多耐药菌株的基因编辑提供了一种新的方法。Red homologous recombination is an important tool for gene editing in Escherichia coli.PKD46,pKD3/4 and pCP20 are commonly used engineering plasmids in this system,which use ampicillin,kanamycin and chloramphenicol as screening markers.When irp2 gene knockout was carried out on a pathogenic Escherichia coli isolated from diarrhea Saba piglets,it was found that the strain was insensitive to ampicillin,chloromycetin and many other kinds of antibiotics,making it hard to do the screening because it was a multidrug-resistant strain.To solve this problem,the target fragment carrying bleomycin resistance and the recombinant plasmid pCas-FLP carrying FLP enzyme were constructed,and the irp2 gene of Escherichia coli A/10 were successfully achieved.The results not only provide a basis for further study of the pathogenic mechanism of irp2 gene,but also provide a new method for gene editing of multidrug-resistant bacteria.

关 键 词：RED同源重组 多耐药 irp2基因缺失 

分 类 号：S852.62[农业科学—基础兽医学]