论文部分内容阅读
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。
Objective To investigate the relationship between the alteration of cell membrane permeability caused by ompC and ompF in outer membrane and the formation of drug resistance of Escherichia coli (ECO). Methods The ompC gene sequences and the ompF, ompA, fepA, cirA, chuA and other gene sequences were amplified by PCR. The PCR products were recovered and passivated and sequenced. The target membrane was recovered by SDS-PAGE electrophoresis Protein bands were trypsinized, analyzed for protein composition by mass spectrometry and subjected to protein sequence determination. Results SDS-PAGE electrophoresis and mass spectrometry analysis showed that all 7 Escherichia coli strains had corresponding ompC and ompA expression without membrane ompF and 6 and 7 strains with ompC and ompA expression But there was expression of iron transport protein chuA, cirAf and epA. The gene amplification showed that all the 7 strains of Escherichia coli had ompF products. The strains 6 and 7 with stronger resistance also had the amplification of fhuA and cirA Product, but the relative molecular mass of the product fhuA was less than that of ECO-k12 (54 bp less). Conclusion The continuous use of antibacterial drugs will lead to low or no expression of bacterial ompF, and the alteration of the amino acid sequence of ompC in the outer membrane will affect the expression of the corresponding proteins and decrease the permeability of the cell membrane, thereby increasing the resistance of the bacteria to antibacterials Highly resistant Escherichia coli with compensatory expression of iron transport protein chuA, cirA, fepA, to maintain bacterial metabolism, maintain its high level of drug resistance.