【摘要】 目的研究外膜孔道蛋白ompC、ompF引起细胞膜通透性改变与大肠埃希菌(ECO)对抗菌药物形成耐药的相关性。方法设计复合引物对ompC全基因序列和ompF、ompA、fepA、cirA、chuA等基因序列进行扩增,回收PCR产物、钝化后测序;菌体外膜蛋白进行SDS-PAGE电泳,回收的目标膜蛋白条带经胰蛋白酶消化,采用质谱仪分析蛋白成分并进行蛋白序列的测定。结果SDS-PAGE电泳图谱和质谱分析显示,7株大肠埃希菌均有相应的外膜孔道蛋白ompC和ompA表达,无外膜孔道蛋白ompF表达;第6、7菌株的孔道蛋白ompC和ompA表达量明显减少,但有铁运输蛋白chuA、cirAf、epA的表达;基因扩增显示7株大肠埃希菌均有ompF产物,耐药性较强的第6、7菌株还有fhuA和cirA扩增产物,但产物fhuA的相对分子质量小于ECO-k12(少54 bp)。结论持续使用抗菌药物将导致细菌外膜孔蛋白ompF低表达或不表达,外膜孔蛋白ompC氨基酸序列的改变影响相应蛋白的表达,使细胞膜通透性降低,从而增加细菌对抗菌药物的耐药性;高度耐药的大肠埃希菌伴随代偿性铁运输蛋白chuA、cirA、fepA的表达,维持细菌的新陈代谢,保持其高水平耐药性。更多还原

【Abstract】 OBJECTIVE To identify and analyze whether the mutations in porins ompC and ompF result in a decrease in outer membrane permeability,and resistence to a number of chemically unrelated antibiotics.METHODS The ompC and ompF genes were sequenced using primers ompC6F,ompC2R,ompC8F and ompC4R.Primers were designed to correspond to the protein regions conserved between K-12(U00096) and ompC from the isolates,which were identified by the mass spectrometry analysis of tryptic fragments derived from ompC protein separated by SDS-PAGE of outer membrane extracts.RESULTS Analysis of outer membrane proteins by SDS-PAGE and peptide mass fingerprinting of bands reveals the presence of ompA and ompC,but not ompF.PCR analysis confirmed the presence of the ompF gene,but no evidence for its expression was obtained.It was notable that exhibited reduced levels of ompC and ompA in the last two isolates(6 and 7).Analysis of the PCR products showed that the fhuA PCR product was shorter for the clinical isolates than for Escherichia coli K-12(54bp).CONCLUSIONS Preliminary data suggest that changes in the amino acid sequence contribute to antibiotic resistance via the modulation of ompC activity.更多还原