| 沈靖,邓大君,柯杨,张建中.幽门螺杆菌阿莫西林耐药株缺乏青霉素结合蛋白基因外突变特征[J].Chinese journal of Epidemiology,2008,29(2):166-172 |
| 幽门螺杆菌阿莫西林耐药株缺乏青霉素结合蛋白基因外突变特征 |
| Detection of point mutation in an in vitro-selected amoxicillin-resistant strain of Helicobacter pylori |
| Received:November 08, 2007 Revised:August 06, 2012 |
| DOI： |
| KeyWord: 幽门螺杆菌 阿莫西林 耐药性 点突变 蛋白质 |
| English Key Word: Helicobacter pylori Amoxicillin Resistance Point mutation Proteomics |
| FundProject:北京市自然科学基金资助项目(7002008);国家"863"高技术研究发展计划课题资助项目(2001AA21516102) |
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| Abstract: |
| 目的 研究实验室诱导的耐阿莫西林(AMO)幽门螺杆菌(H.pylori)的青霉素结合蛋白基因(pbp)突变情况,探讨pbp基因突变与耐药性形成的关系,比较AMO耐药菌株和敏感菌株的蛋白表达谱,为筛选与H.pylori耐药相关的蛋白提供线索.方法 体外诱导敏感菌株H.pylori 26695产生AMO耐药,测定耐药菌株5个PBP的全基因的点突变情况;同时运用蛋白质组学技术,比较AMO耐药菌株和敏感菌株的蛋白表达谱.结果 (1)体外诱导获得MIC为8 μg/ml的耐药菌1株(AMOr),其耐药表型经-80℃冻存或在不含AMO的培养基上多次传代后会丧失;(2)AMOr全部待测序列和出发菌株26695相应的靶序列完全相同,未检出基因点突变等结构变异;(3)对26695和AMOr的蛋白表达图谱进行比较发现:11个蛋白斑点在表达量上有显著变化.结论 H.pylori AMO耐药性的形成主要是一种不稳定的表型变化,可能不是由pbp基因结构变异所致.实验中耐药株和敏感株差异表达的蛋白在H.pylori的耐药形成过程中发挥着怎样的作用还有待进一步研究. |
| English Abstract: |
| objective To investigate the relationship between point mutation of penicillin-binding protein gene (pbp)and amoxicillin resistance (AMO`)of Helicobacter pylori(H.pylori)as well as to compare the protein profiles under proteomic technology to get the candidate resistance-related proteins.Methods(1)AMO` strains were selected from the sensitive H.pylori strain 26695 by serial passage technique in vitro.(2)Point mutations of five putative resistance genes(HP0597, HP1565, HP1542, HP1556, and HP0160) were analyzed by denaturing high-performance liquid chromatography (DHPLC) and DNA sequencing.(3) Proteins samples were separated by two-dimensional electrophoresis (2-DE).Protein profiles were compared between the AMO` strain obtained in vitro and its sensitive parent strain 26695.Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) was performed to identify the proteins of interest.The proteins were searched by software MASCOT and identified by peptide fingerprint map using the program MS-FIT of Protein Prospect.Results(1)An AMO` strain (MIC 8 ug/ml) was obtained.Complete loss of the resistant phenotype was observed after cultivation in the absence of AMO or storage at-80'C.(2)DHPLC and Sequencing result showed no point mutations in five pbp genes in the AMO` strain when compared with the corresponding PCR products from its parent strain 26695.(3)Protein profiling showed that eleven protein spots were differently expressed between 26695 and the AMO' strain.Of these protein spots in the AMO` strain, two new spots (Spot 1 and Spot 2) were observed with one (Spot 3) was up-regulated three-fold and the remained ones (Spot 4-11)were down-regulated.Conclusion AMO resistance of H.pylori might be resulted from unstable phenotype change rather than point mutations of pbp genes.These differentially regulated proteins in AMO' strain might play a role in development of resistance to AMO in H.pylori. |
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