运用融合PCR技术敲除光滑念珠菌FLO8基因以及其对EPA黏附素家族表达的影响

Knocking out FLO8 gene of Candida glabrata and its effect on EPA family

赵珺涛 ¹袁捷 ²刘锦燕 ²陈柯志 ¹项明洁¹

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作者信息

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

目的:构建光滑念珠菌FLO8基因敲除株,并分析FLO8敲除对光滑念珠菌上皮黏附素(epithelial adhe-sin,EPA)家族表达的影响.方法:使用融合PCR技术,以光滑念珠菌ATCC2001菌株基因组DNA、带有筛选标记诺尔斯菌素抗性基因(NAT)的质粒DNA为模板,构建敲除组件.采用醋酸锂转染法将敲除组件转染入ATCC2001中,从而获得flo8△菌株.使用实时荧光定量PCR检测菌株中EPA1、EPA6和EPA7基因的表达.结果:获得光滑念珠菌FLO8基因敲除株flo8△,该菌株的EPA1、EPA6和EPA7基因表达水平对明显低于ATCC2001株(P均<0.001).结论:此法可便捷、有效地构建光滑念珠菌基因敲除菌株.敲除FLO8基因后,光滑念珠菌的EPA家族表达降低,为进一步研究光滑念珠菌毒力机制奠定基础.

Abstract

Objective To construct a FLO8 gene knockout strain of Candida glabrata and analyze the effect of FLO8 knockout on the expression of EPA family in Candida glabrata. Methods By fusion PCR technology,the knockout components were constructed with the genomic DNA of Candida glabrata ATCC2001 strain and the plasmid DNA with screening marker NAT as templates. The knockout components were transfected into Candida glabrataATCC2001 by lithium acetate transfection method to obtainflo8△strain. The expression of EPA1,EPA6 and EPA7 genes were detected by real-time quantitative PCR. Results The FLO8 gene knockout strain of Candida glabrata was efficiently constructed. The expression levels of EPA1,EPA6 and EPA7genes in flo8△strain were significantly lower than those in ATCC2001 strain (all P<0.001). Conclusions The gene knockout method permits rapid,easy and highly efficient generation of homozygous knockout mutations in Candida glabrata. The knockout of FLO8 gene reduced the expression of EPA family in Candida glabrata,which laid a foundation for further study on its virulence mechanism.

关键词

光滑念珠菌/基因敲除/黏附素/FLO8基因/上皮黏附素家族

Key words

Candida glabrata/Gene knockout/Adhesin/FLO8 gene/Epithelial adhesin family

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出版年

2024

诊断学理论与实践

上海交通大学医学院附属瑞金医院

诊断学理论与实践

CSTPCD

影响因子：0.413

ISSN：1671-2870

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