关键词: 基因敲除, 同源重组, 肺炎克雷伯菌

Abstract: 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 bla_KPC-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 bla_KPC-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 bla_{KPC - 2} gene and hygromycin resistance gene, and drug sensitive test was used to confirm the recombination. Results The bla_KPC-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.

Key words: Gene knockout, Homologous recombination, Klebsiella pneumonia