差异显示PCR技术在念珠菌基因表达研究中的应用

目的:探讨差异显示PCR技术(DD-PCR)在念珠菌基因表达研究中的方法学要点.方法:将临床阴道来源的白色念珠菌氟康唑敏感菌株435体外诱导氟康唑耐药性,于诱导80d得到耐药子代435-2,采用长引物差异扩增、银染显示差异条带、反式点杂交预筛选及非放射性法标记探针的改良的DD-PCR,比较435与435-2在有药及无药培养基中的表达差异.结果:在435-2含药培养组中得到较435-2、435无药培养组表达明显增强的片段N2、N3、N12,分别与数据库中白色念珠菌的醇脱氢酶基因ADH1、拓扑异构酶基因TOP2及多药耐药基因CDR1基因有高度同源性;半定量RT-PCR中证实了ADH1、CDR1基因在耐药株中的高表达.结论:ADH1、CDR1基因的高表达与白色念珠菌氟康唑耐药性形成相关,ADH1可能是新的耐药基因;改良的DD-PCR技术降低了假阳性率的产生,使确证步骤更加便捷,在念珠菌研究领域有很好的应用前景.

Application of differential display-PCR technique in fluconazole-resistance gene expression of Candida

OBJECTIVE: To investigate the application of differential display-2PCR(DD-PCR) in research on gene expression of Candida. METHODS: Resistance to fluconazole was induced in a Candida albicans isolate 435 from vagina by culturing in YEPD broth with increasing fluconazole concentration in vitro, and the resistant isolate 435-2 (MIC=128 microg/ml ) was obtained after 80 days of incubation. Comparisons between 435 and 435-2 either in fluconazole-containing medium or in drug-free medium were performed with the modified DD-PCR including amplification with long primers, silver staining, reverse dot blot and non-radiographic labeling techniques. RESULTS: Three differential displayed bands were found which showed high homology to alcohol dehydrogenase 1 (ADH1), TOP2 and CDR1, respectively. The up-regulating expression of ADH1 and CDR1 associated with fluconazole resistance was further identified by RT-PCR. CONCLUSION: The up-regulating expression of ADH1 and CDR1 was associated with fluconazole resistance in Candida albicans, ADH1 might be a candidate of novel fluconazole resistant gene.