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宋春花, 王颖芳, 郗园林, 段广才. 志贺菌诱导耐多药相关基因抑制性消减杂交法筛选[J]. 中国公共卫生, 2013, 29(3): 354-357. DOI: 10.11847/zgggws2013-29-03-14
引用本文: 宋春花, 王颖芳, 郗园林, 段广才. 志贺菌诱导耐多药相关基因抑制性消减杂交法筛选[J]. 中国公共卫生, 2013, 29(3): 354-357. DOI: 10.11847/zgggws2013-29-03-14
SONG Chun-hua, WANG Ying-fang, XI Yuan-lin.et al, . Screening for anti-multidrug associated genes of Shigella flexneri with suppression subtractive hybridization[J]. Chinese Journal of Public Health, 2013, 29(3): 354-357. DOI: 10.11847/zgggws2013-29-03-14
Citation: SONG Chun-hua, WANG Ying-fang, XI Yuan-lin.et al, . Screening for anti-multidrug associated genes of Shigella flexneri with suppression subtractive hybridization[J]. Chinese Journal of Public Health, 2013, 29(3): 354-357. DOI: 10.11847/zgggws2013-29-03-14

志贺菌诱导耐多药相关基因抑制性消减杂交法筛选

Screening for anti-multidrug associated genes of Shigella flexneri with suppression subtractive hybridization

  • 摘要: 目的应用抑制性消减杂交技术研究志贺菌诱导耐多药株与其敏感株之间差异基因,筛选耐多药相关基因,探讨基因表达差异与志贺菌诱导耐多药的相关性。方法以志贺菌诱导耐多药株DNA为检测子(tester),以其敏感株DNA为驱赶子(driver),构建DNA消减文库,采用PCR鉴定及斑点杂交进行筛选,并随机挑取阳性克隆测序,所得结果在GenBank中做同源性比较分析。结果成功构建了志贺菌诱导耐多药株的特异性DNA消减杂交文库;选取部分阳性克隆进行杂交筛选,获得12个诱导耐多药差异片段,对其中6个基因片段经克隆测序并与GenBank数据库进行初步比对,3个未知基因可能为新基因,3个已知基因分别为16S rRNA 核糖体基因、预测的rep蛋白质(解螺旋酶)、位点特异性Ⅰ型脱氧核糖核酸酶(Hsds)等相关基因。结论志贺菌耐多药是多基因参与的复杂过程,筛选的新基因片段为进一步克隆其全长基因进而研究其功能打下基础。

     

    Abstract: ObjectiveTo investigate the differential genes between the induced anti-multidrug strain and sensitive strain of Shigella flexneri with suppression subtractive hybridization(SSH) and to screen the genes related to multidrug resistance and to analyze its relationship with induced drug-resistance.MethodsThe induced anti-multidrug subtractive DNA library was constructed from the DNA of anti-multidrug strain(tester) and sensitive strain of Shigella flexneri(driver).The parts of positive clones in the library were identified by PCR,screened by dot blotting hybridization,and then sequenced and analyzed with the BLAST software in GenBank.ResultsThe induced anti-multidrug subtractive DNA library was constructed successfully.A total of 12 induced multidrug resistant differential fragments were acquired,in which,6 fragments were sequenced and compared with the GenBank Database,3 fragments failed to match with the homologous genes in Genbank,while the other 3 fragments had a high similarity to known genes(16S rRNA gene,hypothetical Rep protein,and type I site-specific deoxyribonuclease).ConclusionAnti-multidrug of Shigella flexneri is a multi-genes involved in a complex process and the screened novel gene fragments could provide basis for further cloning of full-length genes.

     

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