利用Red重组系统敲除鲍曼不动杆菌asa A基因  

Deletion of asaA gene in Acinetobacter baumannii by Red recombination system

作　　者：李磊 王一诺 邹清华[1] LI Lei;WANG Yi-nuo;ZOU Qing-hua(Department of Microbiology, School of Basic Medical Sciences, Peking University, Beijing 10019, China)

机构地区：[1]北京大学基础医学院病原生物学系,北京100191 [2]全国医学教育发展中心,北京100191

出　　处：《微生物学免疫学进展》2019年第1期14-18,共5页Progress In Microbiology and Immunology

基　　金：国家自然科学基金(81572041)

摘　　要：目的利用Red重组系统敲除鲍曼不动杆菌ATCC 17978的asaA。方法设计上下游引物中包含asaA的同源序列,并以pKD4质粒为模板,扩增含有卡那霉素抗性基因的DNA片段。将该片段转化于表达重组酶的17978感受态细胞中,在卡那霉素筛选压力下,得到经两次同源双交换的具有卡那霉素基因标记的突变菌株。随后,在重组酶的作用下将抗性基因去除,最终得到无抗性基因标记的突变菌株ΔasaA。结果通过该重组系统,首先将卡那霉素抗性基因的DNA片段替换了基因组中asaA的DNA片段,然后将卡那霉素抗性基因的DNA片段消除,最终获得了asaA缺失的突变体。结论通过Red重组系统为鲍曼不动杆菌中其他基因的缺失突变提供方法与思路。Objective To construct an asaA gene deletion mutant for Acinetobacter baumannii ATCC 17978 with red recombination system. Methods The homologous fragments including homologous regions which are similar to asaA gene upstream and downstream sequence, with pKD4 plasmid as a template, respectively, and kanamycin cassette were amplified. The PCR products were introduced into ATCC 17978 competent cells by electroporation, under the pressure of kanamycin, homologous recombination occurred twice between the fragments and genome of host strain, by using the FLP recombinase, to prepare the gene deficient mutant without antibiotic marker. Results The asaA deletion mutant of ATCC 17978 was successfully constructed by red recombination system, in which the antibiotic resistance marker was removed. and resulted in a single gene deficient mutant ΔasaA without antibiotic marker. Conclusion This performed deletion provided the clue for knock-out of other genes in Acinetobacter baumannii .

关 键 词：鲍曼不动杆菌 基因敲除 Ⅵ型分泌系统 asaA 

分 类 号：Q78[生物学—分子生物学]