luxS基因敲除临床分离大肠杆菌株生物膜形成能力的变化

Effect of luxS gene on biofilm formation in knocked-out clinically isolated Escherichia coli

目的探讨LuxS基因对临床分离大肠杆菌生物膜形成能力的影响。方法以临床分离大肠杆菌S17为研究对象,PCR分别扩增S17 LuxS基因的上下游序列和质粒pEGFP-N1的卡那抗性基因,分别连入T载体pAT2,再通过酶切连接的方法分别将LuxS基因的上、下游序列连接入pAT2-kana质粒,构建同源重组质粒pAT2-△luxS,同源重组质粒转化大肠杆菌DH5α扩增后,提取质粒后双酶切获得同源重组片段,将同源重组片断电转入感受态S17,通过同源重组构建LuxS基因缺失的S17,96孔板结晶紫染色法分析LuxS基因缺失株生物膜形成能力的变化。结果 LuxS基因缺失株基因组PCR扩增出1678的片断,测序鉴定正确,成功构建临床分离大肠杆菌S17 LuxS基因缺失株,S17与S17LuxS基因缺失株形成生物膜的微孔板法半定量OD值的结果分别为(1.51±0.09)和(0.43±0.13)。结论 LuxS基因对细菌生物膜的形成具有重要调控作用。

Objective To study the effect of luxS gene on biofilm formation in knocked-out Escherichia coli（E.coli）.Methods Clinically isolated E.coli S17 was used as the target in this study.The upstream and downstream sequences of luxS,and kana-resistance gene of pEGFP-N1 were amplified by PCR,cloned into T plasmid pAT2 vector and pAT2-kana.Homologous recombinant plasmids pAT2-△luxS were then constructed.After the pAT2-△luxS were transformed into E.coli and amplified,fragments of homologous recombinant plasmids were obtained by restricted enzyme reaction and transformed to E.coli S17.Effect of luxS gene on biofilm formation in knocked-out E.coli in 96-well plates was analyzed with crystal violet staining.Results A total of 1678 luxS fragments were amplified by PCR with correct sequences.A E.coli S17 strain without luxS gene was established with an OD of 1.51±0.09 and 0.43±0.13,respectively.Conclusion The luxS gene plays an important role in regulation of biofilm formation in E.coli.