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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。
OBJECTIVE: To study the differential gene between multidrug-resistant strains and its sensitive strains induced by Shigella and to screen multidrug resistance-related genes by suppression subtractive hybridization to explore the correlation between gene expression differences and multidrug resistance induced by Shigella. Methods DNA was extracted from Shigella multidrug-resistant strains using tester DNA as driver and DNA subtractive library was constructed. The PCR products were identified by dot blot hybridization and positive clones Sequencing, the results obtained in GenBank comparative analysis of homology. Results The specific DNA subtractive hybridization library of Shigella multidrug-resistant strains was constructed successfully. Some positive clones were screened by hybridization and 12 differentially-differentially expressed fragments were obtained. Six of them were cloned and sequenced, and compared with GenBank Three unknown genes may be novel genes, three known genes are 16S rRNA ribosomal genes, predicted rep proteins (helicases), site-specific type I deoxyribonucleases (Hsds ) And other related genes. Conclusion Shigella multidrug resistance is a complicated process involving multiple genes. The new gene fragments screened further lay the foundation for further cloning of its full length gene and its function.