CAJ | 학술논문

目的：建立敲除临床肺炎克雷伯菌耐药菌株质粒上的耐药基因的方法。方法聚合酶链反应（PCR）分别扩增试验菌株待敲除目的基因blaKPC-2的上下游片段及质粒pMQ300的潮霉素耐药基因片段，采用重叠延伸基因扩增（SOE-PCR）技术构建融合片段，以耐阿普霉素的λred质粒pKOBEG-Apr为介导，同源重组敲除临床肺炎克雷伯菌耐药菌株的blaKPC-2基因。用含潮霉素和阿普霉素的LB培养基筛选重组子，用PCR和逆转录PCR扩增检测blaKPC-2基因、潮霉素耐药基因及药物敏感性验证重组子。结果不同多位点序列分析（MLST）型别的2株临床肺炎克雷伯菌耐药菌株的blaKPC-2基因得以敲除。结论λred同源重组法可以可靠敲除临床肺炎克雷伯菌耐药菌株质粒上的基因。
목적：건립고제림상폐염극뢰백균내약균주질립상적내약기인적방법。방법취합매련반응（PCR）분별확증시험균주대고제목적기인blaKPC-2적상하유편단급질립pMQ300적조매소내약기인편단，채용중첩연신기인확증（SOE-PCR）기술구건융합편단，이내아보매소적λred질립pKOBEG-Apr위개도，동원중조고제림상폐염극뢰백균내약균주적blaKPC-2기인。용함조매소화아보매소적LB배양기사선중조자，용PCR화역전록PCR확증검측blaKPC-2기인、조매소내약기인급약물민감성험증중조자。결과불동다위점서렬분석（MLST）형별적2주림상폐염극뢰백균내약균주적blaKPC-2기인득이고제。결론λred동원중조법가이가고고제림상폐염극뢰백균내약균주질립상적기인。
Objective To establish a method of knocking out drug-resistant genes on plasmid contained by clinical Klebsiella pneumonia drug-resistant isolates.Methods Polymerase chain reaction (PCR)was used to amplify the upstream and downstream fragments of target gene blaKPC-2 of the test isolates and hygromycin resistance gene fragments on plasmid pMQ300,respectively.Gene splicing by overlap extension polymerase chain reaction(SOE-PCR)was used to construct the fusion fragments,and apramycin resistance lambda red plasmid pKOBEG-Apr was used as mediated, homologous recombination knockout blaKPC-2 gene in clinical Klebsiella pneumonia drug-resistant isolates.LB medium containing hygromycin and apramycin was used to screen recombination,and PCR and reverse transcription-polymerase chain reaction (RT-PCR)amplification were used to detect blaKPC-2 gene and hygromycin resistance gene,and drug sensitive test was used to confirm the recombination.Results The blaKPC-2 gene was successfully knocked out in 2 clinical Klebsiella pneumonia drug-resistant isolates with different multilocus sequence typing(MLST).Conclusions Lambda red homologous recombination method can be used to knock out clinical Klebsiella pneumonia drug-resistant isolate plasmid gene reliably.