基于RecAb系统的鲍曼不动杆菌Omp33-36缺陷株的构建及鉴定  

Construction and identification of an Acinetobacter baumannii Omp33-36 defective strain based on an RecAb system

作　　者：王慧璇 王胜军[1] 程建军 蔡伟 许化溪[1] WANG Hui-xuan;WANG Sheng-jun;CHENG Jiang-jun;CAI Wei;XU Hua-xi(Department of Immunology Laboratory,Institute of Laboratory Medicine,Jiangsu University,Zhenjiang,Jiangsu 212013,China)

机构地区：[1]江苏大学检验医学研究所免疫研究室,江苏镇江212013

出　　处：《中国病原生物学杂志》2020年第10期1117-1123,1130,共8页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81771756)。

摘　　要：目的构建鲍曼不动杆菌ATCC17978外膜蛋白Omp33-36基因缺陷株。方法pAT04质粒电穿孔鲍曼不动杆菌ATCC 17978所得ATCC17978-pAT04经IPTG诱导后用甘油法制备电穿孔用感受态细菌。以ATCC 17978基因组为模板PCR扩增Omp33-36的上下游基因,以pKD4质粒为模板扩增卡那霉素抗性基因1477 bp片段,并以上述3种PCR产物割胶回收的混合物作为融合PCR模板,扩增Omp33-36同源臂卡那霉素抗性盒DNA片段(2111 bp);以此融合PCR产物为模板巢式PCR扩增同源臂卡那霉素抗性盒DNA片段(1977 bp)并电穿孔至感受态ATCC17978-pAT04;经卡那霉素筛选及PCR鉴定得到同源重组菌株ΔOmp::FRT-kanR(pAT04),去除pAT04质粒,得到菌株ΔOmp::FRT-kanR。将pAT03质粒电穿孔感受态Omp::FRT-kanR细菌,得到ΔOmp::FRT-kanR(pAT03)菌株,再经IPTG诱导表达FLP重组酶去除卡那霉素抗性基因,得到ΔOmp::FRT(pAT03)菌株,进一步去除pAT03质粒后得到敲除Omp33-36基因的目的菌株ΔOmp::FRT并测序鉴定。超速离心法提取细菌OMVs并进行SDS-PAGE;E-test测定ATCC17978野生株和Omp33-36敲除株对碳青霉烯类的最低抑菌浓度(MIC)值。结果成功构建鲍曼不动杆菌Omp33-36敲除株ΔOmp::FRT,测序表明敲除的Omp33-36基因座位被FRT序列及卡那霉素扩增引物序列所替代。从Omp33-36敲除株ΔOmp::FRT提取的OMVs经SDS-PAGE电泳显示,与野生株相比约33 ku蛋白组分几尽缺失。敲除株ΔOmp::FRT对美罗培南的MIC值高于野生株。结论利用RecAb系统成功敲除鲍曼不动杆菌外膜蛋白Omp33-36基因,建立了Omp33-36缺陷株,其对美罗培南的耐受性增高。Objective To use an RecAb system to construct a mutant strain of Acinetobacter baumannii ATCC17978 with deletion of the outer membrane protein Omp33-36 gene.Methods The plasmid pAT04 was transformed into A.baumannii ATCC 17978 by electroporation,and the resulting strain was designated ATCC17978-pAT04.Expression of ATCC17978-pAT04 was induced with IPTG,and electroporation-competent ATCC17978-pAT04 was prepared using the glycerol method.Genes upstream and downstream of Omp33-36 were amplified with PCR using the ATCC 17978 genome as a template,and a kanamycin resistance gene fragment 1,477 bp in length was amplified using pKD4 plasmid as a template.A mixture of the above three PCR products above served as a fusion PCR template with which to amplify the homologous arms of Omp33-36 and a kanamycin resistance cassette DNA fragment 2,111 bp in length.The fusion PCR products were used as a template in nest PCR to amplify a linear DNA fragment 1,977 bp in length containing the homologous arms of Omp33-36 and kanamycin resistance cassette DNA.These were introduced into ATCC17978-pAT04 via electroporation.The recombinant strainΔOmp::FRT-kan^R(pAT04)expressing a kanamycin resistance gene was selected using kanamycin agar and verified via amplification with PCR.The strainΔOmp::FRT-kan^R was obtained by removing the pAT04 plasmid fromΔOmp::FRT-kan^R(pAT04).TheΔOmp::FRT-kan^R(pAT03)strain was obtained by electroporation of the pAT03 plasmid intoΔOmp::FRT-kan^R bacteria.The kanamycin resistance gene was removed using FLP recombinase,expression was induced with IPTG,and theΔOmp::FRT(pAT03)strain was obtained.After removal of the pAT03 plasmid,the Omp33-36 deletion strainΔOmp::FRT was obtained and further identified using sequencing.Bacterial OMVs were extracted using ultracentrifugation and subjected to SDS-PAGE protein electrophoresis.An E-test was used to determine the minimum inhibitory concentration(MIC)of carbapenems with respect to the wild-type ATCC 17978 strain and the Omp33-36 knockout strain.Results The mutant st

关 键 词：鲍曼不动杆菌ATCC17978 RecAb系统 Omp33-36基因 同源重组 基因敲除 

分 类 号：R378[医药卫生—病原生物学]