单增李斯特菌FosX缺失突变菌的构建  

Construction of Listeria monocytogenes FosX deletion mutant

作　　者：韩蕾[1] 韩少山 徐纪茹[1] 

机构地区：[1]西安交通大学医学院免疫学与病原生物学系,陕西西安710061 [2]第一附属医院肝胆外科,陕西西安710061

出　　处：《西安交通大学学报（医学版）》2013年第3期336-340,共5页Journal of Xi’an Jiaotong University（Medical Sciences）

摘　　要：目的构建单增李斯特菌FosX缺失型突变菌株。方法设计并合成特异性引物，PCR扩增josX基因上、下游片段DNA（1＋2）和DNA（3＋4）．并通过酶切和连接将两条目的片段先、后克隆入载体pMADh构建F1的质粒pMAD-DNA（1＋4）。随后将目的质粒电穿孔转化入感受态单增李斯特菌中．通过抗乍素压力和温度压力筛选出突变中间体，PCR鉴定阳性克隆。最后在无抗生素压力的条件下传代突变中间体，PCR扩增鉴定突变菌。用Etest法检测构建的单增李斯特菌FosX缺失突变菌及其亲代菌株的磷霉素最小抑菌浓度（MIC）。结果成功构建了单增李斯特菌FosX缺失突变菌。药敏结果显示．缺失FosX后磷镱素MIC值显著下降，单增李斯特菌对磷霉素的敏感性大火增加。结论FosX缺失突变菌构建成功，并存功能学上证实了FosX的耐药因子作用，为进一步研究FosX引发的耐药作用平和Hpt及PrfA介导的敏感作用之间的相互关系奠定了实验基础。Objective To construct FosX dclction mutant in Listeria monocytogenes strain. Methods With specific primers. PCR was performed to amplify the upper and lowcr regions of fosX gcnc. Thc upper and lower regions as weld as plasmid pMAD wcrc digested by restriction cndonucleasc, followed by ligation using T4 DNA ligasc to construct the target plasmid pMAD-DNA （1 ＋ 4）. After the target plasmid was elcctroporatcd into L. monocytogenes, the intcrmcdiate construction was selected by antibiotic and temperature pressures. Final mutant was investigated by PCR after passaging without antibiotic pressure. Fosfomycin susceptibility test was carried out using Etcst strips. Results The L, rnonocytogenes FosX deletion mutant was constructed succcssfully. The fosfomycin MIC of FosX deletion mutant decreased dramatically compared to that of thc parental strain, indicating the improved fosfomycin susceptibility. Conclusion The successfully constructcd L. monocytogenes FosX deletion mutant vcrificd the function of FosX in fosfomycin resistance and provided empirical basis for investigating the essential fosfomycin susccptibility of L. monocytogenes based on both FosX and Hpt as well as PrfA.

关 键 词：单增李斯特菌 FosX 缺失突变 

分 类 号：Q933[生物学—微生物学]