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- 2018
肺炎克雷伯菌果糖磷酸转移酶系统EⅡC编码基因frwC缺失株的构建及表型分析
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Abstract:
摘要：目的 利用同源重组技术构建肺炎克雷伯菌果糖磷酸转移酶系统(PTS系统)EⅡC编码基因frwC无痕缺失株，并构建回补株，探讨frwC基因对肺炎克雷伯菌生长、超黏表型及毒力的影响。 方法 利用自杀载体pKO3-Km质粒构建frwC基因缺失株，同时扩增包含frwC基因编码区、启动区及转录终止区的基因片段，克隆至pGEM-T-easy质粒电转至缺失株构建frwC基因回补株。通过生长曲线测定、高速离心实验、小鼠毒力实验分析frwC基因对肺炎克雷伯菌生长、超黏表型及毒力的影响。结果 成功构建了肺炎克雷伯菌frwC基因缺失株与回补株，发现frwC基因敲除后细菌生长速度下降、黏性增加、细菌毒力减弱。结论 肺炎克雷伯菌果糖磷酸转移酶系统frwC基因编码蛋白EⅡC影响细菌的生成、超黏表型及毒力。
ABSTRACT: Objective To construct the frwC gene deletion mutant by homologous recombination and the frwC complementation strain of Klebsiella pneumoniae NTUH-K2044 and analysis of its phenotypes. Methods The frwC gene deletion mutant was constructed by using suicide vector pKO3-Km, and then the gene fragment including frwC coding region, promoter area and transcription termination area were amplified and cloned into pGEM-T-easy plasmid to acquire frwC complementation strain. The phenotypes of the wild-type strain, the frwC mutant, and the complemented mutant were characterized. Results The frwC depletion mutant and complementation strain were constructed successfully. The growth rate and virulence of frwC mutant were decreased, but the viscosity was increased. Conclusion K. pneumoniae frwC was required for in vitro growth, capsular polysaccharide biosynthesis, and full virulence in a Klebsiella pneumoniae strain
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