光滑念珠菌EPA1和EPA6基因敲除及其对生物膜形成的影响

doi:10.16150/j.1671-2870.2026.01.011

诊断学理论与实践 ›› 2026, Vol. 25 ›› Issue (01): 78-84.doi: 10.16150/j.1671-2870.2026.01.011

光滑念珠菌EPA1和EPA6基因敲除及其对生物膜形成的影响

刘锦燕¹, 宋怡慧^2a^,^*, 王鲁灵^2b, 陈柯志³, 赵珺涛^2b, 吕埂^2b, 项明洁¹^,^2b()

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 a.上海市高血压研究所，b.检验科，上海 200025
3.四川大学华西医院实验医学科，四川大学华西医院临床检验医学研究中心，四川省医学检验临床医学研究中心，成都 610041

收稿日期:2025-09-20 修回日期:2025-11-04 接受日期:2025-11-19 出版日期:2026-02-25 发布日期:2026-02-25
通讯作者: 项明洁 E-mail： mjxiang123456@126.com
作者简介:^*：并列第一作者
基金资助:
上海市科委课题(22ZR1439800);上海市卫健委课题(202240205);上海市黄浦区卫健委课题(HLM202303)

Knockout of EPA1 and EPA6 genes in Candida glabrata and their impact on biofilm formation

LIU Jinyan¹, SONG Yihui^2a^,^*, WANG Luling^2b, CHEN Kezhi³, ZHAO Juntao^2b, LV Geng^2b, XIANG Mingjie¹^,^2b()

1. Radioimmunology and Clinical Laboratory, Ruijin Hospital Luwan Branch, Shanghai Jiao Tong University School of Medicine, Shanghai 200020, China
2. The Shanghai Institute of Hypertension, 2b. Department of Clinical Laboratory, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
3. Department of Laboratory Medicine, Clinical Laboratory Medicine Research Center, West China Hospital, Sichuan University, Sichuan Clinical Research Center for Laboratory Medicine, Chendu 610041, China

Received:2025-09-20 Revised:2025-11-04 Accepted:2025-11-19 Published:2026-02-25 Online:2026-02-25

摘要/Abstract

摘要：

目的：构建光滑念珠菌上皮黏附素EPA1和EPA6基因敲除株，并分析基因敲除后对光滑念珠菌生物膜形成的影响。方法：利用融合PCR和同源重组技术，以光滑念珠菌ATCC2001菌株基因组DNA、带有筛选标记潮霉素抗性基因（HyR）的质粒DNA为模板，构建EPA1和EPA6基因敲除组件。采用醋酸锂转染法将敲除组件转染入ATCC2001中，从而获得Δepa1和Δepa6敲除株。采用疏水性实验和生物膜生成实验，观察敲除株毒力表型。结果：与标准株ATCC 2001的疏水性能力（91.5%）比较，本研究获得的光滑念珠菌Δepa1和Δepa6的疏水性能力分别降至61.6%和75.5%（P<0.05）。在30 ℃条件下孵育4 h、16 h和24 h后，Δepa1和Δepa6的生物膜生成能力均显著低于标准株ATCC 2001（P<0.05）。结论：成功构建光滑念珠菌EPA1和EPA6敲除菌株，并证实EPA1、EPA6基因可促进菌株黏附及生物膜形成。

关键词: 光滑念珠菌, 上皮黏附素家族, 基因敲除, 生物膜

Abstract:

Objective To construct Candida glabrata epithelial adhesin EPA1 and EPA6 gene knockout strains and analyze the effects of gene knockout on the biofilm formation of Candida glabrata. Methods Using fusion PCR and homologous recombination techniques, EPA1 and EPA6 gene knockout constructs were generated, with genomic DNA of Candida glabrata strain ATCC 2001 and plasmid DNA carrying the selectable marker hygromycin resistance gene (HyR) as templates. The knockout constructs were transfected into ATCC 2001 using the lithium acetate transfection method to obtain Δepa1 and Δepa6 knockout strains. The virulence phenotypes of the knockout strains were observed through hydrophobicity tests and biofilm formation experiments. Results Compared with the standard strain ATCC 2001, whose hydrophobicity was 91.5%, the hydrophobicity of Δepa1 and Δepa6 obtained in this study decreased to 61.6% and 75.5%, respectively (P<0.05). After incubation at 30℃ for 4, 16, and 24 hours, the biofilm formation abilities of Δepa1 and Δepa6 were significantly lower than those of the standard strain ATCC 2001 (P<0.05). Conclusions The Candida glabrata EPA1 and EPA6 gene knockout strains are successfully constructed, and it is confirmed that the EPA1 and EPA6 genes can enhance strain virulence biofilm formation.

Key words: Candida glabrata, Epithelial adhesin family, Gene knockout, Biofilm

中图分类号:

R446.5

刘锦燕, 宋怡慧, 王鲁灵, 陈柯志, 赵珺涛, 吕埂, 项明洁. 光滑念珠菌EPA1和EPA6基因敲除及其对生物膜形成的影响[J]. 诊断学理论与实践, 2026, 25(01): 78-84.

LIU Jinyan, SONG Yihui, WANG Luling, CHEN Kezhi, ZHAO Juntao, LV Geng, XIANG Mingjie. Knockout of EPA1 and EPA6 genes in Candida glabrata and their impact on biofilm formation[J]. Journal of Diagnostics Concepts & Practice, 2026, 25(01): 78-84.

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参考文献 16

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Primers	Sequences (5′→3′)
EPA1-up-F	GGTACAGAGACGCTACTGCAT
EPA1-up-R	cccggacagccgctaggaggtAAGCTTTGTGAAGGCAAATGTGA
EPA1-down-F	ccgtagcccgatagtcccgagTTCTTGCAGGCGGATCACAT
EPA1-down-R	GAGCGGCATGTTAAGCCTTG
EPA6-up-F	TGCTTTCGTAGATTCAAGCTCAAC
EPA6-up-R	cccggacagccgctaggaggtCACATTTCCTAAGATAGGGTTGTGA
EPA6-down-F	ccgtagcccgatagtcccgagCCACTGCTCAATCGCTTCAC
EPA6-down-F	TCAATGTGCAGAGGATAACTGA
HYR-F	acctcctagcggctgtccgggAACTATGCGGCATCAGAGCA
HYR-R	ctcgggactatcgggctacggTCACTGGCCGTCGTTTTACA
EPA1-in-jd-F	TCGTGTATGGCGAATGGGAG
EPA1-in-jd-R	GAGGGCTGCCCAGGTTTAAT
EPA1-out-jd-F	CCGATGTTCTAAAGATAACGGAGC
EPA1-out-jd-R	TCCTTCGTTTATATACTTCTGTGCC
EPA6-in-jd-F	GAGTCAACTGAGGCACACGA
EPA6-in-jd-R	CCAATGGGGTGTACTTCGCT
EPA6-out-jd-F	TGTACTGACTAGTGGTCATAATCG
EPA6-out-jd-R	ACAGATGCCTTTCTCCAACTAA

光滑念珠菌EPA1和EPA6基因敲除及其对生物膜形成的影响

Knockout of EPA1 and EPA6 genes in Candida glabrata and their impact on biofilm formation

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