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

doi:10.3969/j.issn.1673-8640.2014.07.014

检验医学 ›› 2014, Vol. 29 ›› Issue (7): 750-754.DOI: 10.3969/j.issn.1673-8640.2014.07.014

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

刘锦燕¹，倪培华²，史册¹，魏冰¹，项明洁³

1.上海交通大学医学院附属瑞金医院卢湾分院放免检验科，上海 200020；
2.上海交通大学医学院附属瑞金医院检验系，上海 200025；
3.上海交通大学医学院附属瑞金医院检验科，上海 200020

收稿日期:2013-09-22 出版日期:2014-07-30 发布日期:2014-07-21
通讯作者: 项明洁，联系电话:021-63867643。
作者简介:刘锦燕，女，1981年生，学士，主管技师，主要从事真菌耐药机制和流行病学研究。
基金资助:
上海市科委课题(114119b0500)；上海交通大学医学院课题(09XJ21036)；上海市卫生局课题(2009239)

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

LIU Jinyan¹,NI Peihua²,SHI Ce¹,WEI Bing¹,XIANG Mingjie³

1. Department of Radioimmunology and Laboratory Medicine, Luwan Branch, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200020, China;
2.Faculty of Laboratory Medicine, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China;
3.Department of Clinical Laboratory, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200020, China

Received:2013-09-22 Online:2014-07-30 Published:2014-07-21

摘要/Abstract

摘要：

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。

关键词: 白念珠菌, 氟康唑, ERG11基因, 错义突变, 定点突变技术, 酿酒酵母, 异源性表达

Abstract:

Objective　To verify the correlation of K143Q amino acid substitution in 14α-demethylase and fluconzole resistance to Candida albicans. Methods　The recombinant mutant plasmid with A427C 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 K143Q 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 K143Q contributes to increase the resistance to fluconazole in Candida albicans clinical isolates.

Key words: Candida albicans, Fluconazole, ERG11 gene, Missense mutation, Site-directed mutagenesis, Saccharomyces cerevisiae, Heterologous expression

中图分类号:

R446.5

刘锦燕，倪培华，史册，魏冰，项明洁. 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[J]. 检验医学, 2014, 29(7): 750-754.

LIU Jinyan,NI Peihua,SHI Ce,WEI Bing,XIANG Mingjie. Study on the relationship between K143Q amino acid substitution in 14α-demethylase and fluconazole resistance to Candida albicans[J]. , 2014, 29(7): 750-754.

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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

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