上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

融合PCR结合同源重组技术敲除白色假丝酵母菌FLO8基因

李文静1,2，刘锦燕2，史册1，王影1,2，赵悦1,2，项明洁1,2   

  1. 1.上海交通大学 医学院附属瑞金医院检验科， 上海 200025；2.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
  • 出版日期:2016-03-28 发布日期:2017-06-02
  • 通讯作者: 项明洁, 电子信箱: mjxiang123456@126.com。
  • 作者简介:李文静（1991—），女，硕士生; 电子信箱: 15000028775@163.com。
  • 基金资助:

    上海市科委基金（15ZR1426900）；上海市医学重点专科建设项目（ZK2012A21）；上海市黄浦区优秀青年人才（RCPY1407）

Knock out FLO8 gene in Candida albicans by fusion PCR combined with homologous recombination

LI Wen-jing1,2, LIU Jin-yan2, SHI Ce1, WANG Ying1,2, ZHAO Yue1,2, XIANG Ming-jie1,2   

  1. 1.Department of Clinical Laboratory, Ruijin Hospital，Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China; 2.Radioimmunology and Clinical Laboratory, Luwan Branch, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine,Shanghai 200020, China
  • Online:2016-03-28 Published:2017-06-02
  • Supported by:

    Foundation of Science and Technology Commission of Shanghai Municipality, 15ZR1426900； Key Medical Disciplines Construction Program of Shanghai, ZK2012A21； Excellent Youth of Huangpu District of Shanghai, RCPY1407

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摘要：

目的 运用融合PCR和同源重组技术敲除白色假丝酵母菌FLO8基因，并初步分析敲除菌株药物敏感性(药敏)情况。方法 运用融合PCR将FLO8基因的上下游片段与筛选标记融合在一起构成同源敲除组件，再用高效醋酸锂转染法将敲除组件转染入白色假丝酵母菌SN152，在营养缺陷板上进行筛选，通过2次同源重组敲除FLO8的2条等位基因。采用点板法比较FLO8未敲除株与敲除株药敏情况。结果 成功构建白色假丝酵母菌SN152 FLO8双等位基因缺失株，且耐药性FLO8-/->FLO8＋/->SN152。结论 融合PCR结合同源重组技术能高效、快速构建白色假丝酵母菌FLO8基因缺失株，白色假丝酵母菌FLO8基因与药敏相关，且FLO8拷贝数越少耐药性越强。

关键词: 白色假丝酵母菌, FLO8基因, 基因敲除, 耐药性

Abstract:

Objective To knock out the FLO8 gene in Candida albicans using fusion PCR combined with homologous recombination and analyze drug sensitivity in FLO8 gene knockout strains preliminarily. Methods Upstream and downstream flanking sequences of FLO8 gene were fused with selection markers by fusion PCR to construct homologous knockout fragments, which were then transfected into Candida albicans SN152 by high efficient lithium acetate transfection method. Positive strains were screened on nutritional defect medium and two alleles in FLO8 gene were knocked out by performing the homologous recombination twice. Drug sensitivity was compared between strains with and without FLO8 gene by spot assay. Results The Candida albicans SN152 FLO8-/- double allelic deletion strains were successfully constructed. Drug resistance in FLO8-/- was greater than that in FLO8＋/-, which was greater than that in SN152. Conclusion Fusion PCR combined with homologous recombination can rapidly and efficiently construct Candida albicans FLO8 gene deletion strains. Candida albicans FLO8 gene is associated with drug sensitivity and drug resistance increases with the decrease in copy number.

Key words: Candida albicans, FLO8 gene, gene knockout, drug resistance