白念珠菌14-α脱甲基酶K143Q氨基酸置换与氟康唑耐药形成的相关性研究

Study on the relationship between K143Q amino acid substitution in 14α-demethylase and fluconazole resistance to Candida albicans

目的研究临床耐氟康唑白念珠菌14-α脱甲基酶的K143Q氨基酸置换与氟康唑耐药形成的关系。方法运用体外定点突变技术构建ERG11基因A427C突变型重组质粒,并利用酶切、连接构建YES2/CT酵母表达质粒。通过构建成的表达质粒在酿酒酵母中的异源性表达及体外药物敏感性表型的检测,分析其致K143Q氨基酸置换与白念珠菌对氟康唑产生耐药的关系。结果真菌体外药物敏感性表型检测显示,含氨基酸置换的表达质粒转染于酿酒酵母INVSc1后,氟康唑最低抑菌浓度(MIC)增加16倍。结论 K143Q可增强白念珠菌对氟康唑的耐药性。

Objective To verify the correlation of K143 Q amino acid substitution in 14α-demethylase and fluconzole resistance to Candida albicans. Methods The recombinant mutant plasmid with A427 C in ERG11 gene was firstly constructed by site-directed mutagenesis,and YES2 /CT yeast expression plasmid was established by digestion and ligation technique. The heterologous expression in Saccharomyces cerevisiae and drug susceptibility in vitro were performed to analyze the correlation of K143 Q amino acid substitution with resistance to fluconazole. Results The antifungal drug susceptibility test in vitro showed that the constructed plasmid with amino acid substitution transferred in Saccharomyces cerevisiae INVSc1 contributed to increase in fluconazole minimal inhibitory concentration（ MIC） for 16 times. Conclusions The K143 Q contributes to increase the resistance to fluconazole in Candida albicans clinical isolates.