大肠埃希菌过表达lexA基因对恩诺沙星杀菌力和抗药性突变的影响  

Infl uence on enrofl oxacin bactericidal and resistance mutations by overexpressed lexA gene in Escherichia coli

作　　者：牛卫卫[1,2] 白华[3] 王进文[2] 颜世敢[2] 齐静[2] 骆延波[2] 李璐璐[2] 贾纪美 李靖冉[1,2] 黄娟[1] 单虎[1] 刘玉庆[2] 

机构地区：[1]山东省预防兽医学重点实验室,青岛农业大学,青岛266109 [2]山东省农业科学院畜牧兽医研究所,山东省动物疫病防治与繁育重点实验室,济南250100 [3]山东省畜牧兽医局,济南250022

出　　处：《中国抗生素杂志》2013年第12期942-946,共5页Chinese Journal of Antibiotics

基　　金：国家自然科学基金(81271886);国家科技支撑计划(2011BAD34B03);科技基础性工作专项(SQ2012FY3260033);山东省自然科学基金(ZR2010HQ047)

摘　　要：目的构建过表达SOS阻遏蛋白编码基因lexA的大肠埃希菌菌株,研究SOS应答对恩诺沙星抗药性突变的影响,并建立SOS应答抑制剂筛选平台。方法构建lexA重组表达质粒pET-22b(+)-lexA,转化大肠埃希菌BL21(DE3)构建重组质粒菌株BL21-lexA,用IPTG诱导BL21-lexA过表达lexA,通过SDS-PAGE和Western blot鉴定LexA蛋白,用荧光定量PCR测定SOS应答基因recA和umuC表达变化;然后用恩诺沙星梯度平板测定恩诺沙星对BL21-unmodified(空质粒菌株)和BL21-lexA的抑菌活性和抗药性突变率。结果重组质粒菌株BL21-lexA在IPTG诱导下过表达LexA,抑制recA和umuC表达,提高了大肠埃希菌对恩诺沙星的敏感性,降低了抗药性突变率。结论通过IPTG诱导BL21-lexA过表达LexA蛋白,能构建稳定的SOS应答抑制模型,用于筛选SOS抑制剂,提高恩诺沙星杀菌力和降低抗药性突变。Objective In order to explore the relationship between the SOS response and drug-resistant mutation, the lexA gene, a repressor of the SOS response was overexpressed in Escherichia coli （E. coli） and the influence of overexpression on the bactericidal activity of enrofloxacin （EN） was investigated, and the SOS response inhibitor screening platform was established. Methods Recombinant expression plasmid of pET-22b （＋）-lexA was constructed by inserting the lexA gene into the prokaryotic expression vector pET-22b （＋）, and transformed into the bacterial competent E. coli BL21 （DE3） cells named BL21-lexA strain. The LexA protein was identified through SDS- PAGE and western blot analysis. Meanwhile, the expression of SOS response gene recA and umuC was detected by fluorescence quantitative PCR. Finally, the bactericidal activity and drug resistance mutation rate of BL21-unmodified and BL21-lexA were determined by linear gradient plate. Results The overexpression of LexA of recombinant strain BL21-lexA by IPTG induction inhibited the expression of recA and umuC, and reduced the resistance and its mutation rate of E.coli to enrofloxacin. Conclusion By IPTG inducing BL21-lexA to overexpress LexA protein, the bactericidal action of EN was improved while its drug-resistance mutation was reduced, which established a stable SOS response inhibition model for screening SOS inhibitors.

关 键 词：LEXA SOS应答 大肠埃希菌 恩诺沙星 抗药性突变 质粒过表达 

分 类 号：R378[医药卫生—病原生物学]