白念珠菌SPE1基因高表达菌株构建及鉴定

中国真菌学杂志　 2013, Vol. 8 　Issue (4): 198-201.

白念珠菌SPE1基因高表达菌株构建及鉴定

姚响文, 赵柳娅, 颜钰, 鹿辉, 曹颖瑛, 姜远英

第二军医大学药学院新药研究中心, 上海 200433

收稿日期:2013-05-27 出版日期:2013-08-28 发布日期:2013-08-28
通讯作者: 姜远英,E-mail:jiangyy@smmu.edu.cn E-mail:jiangyy@smmu.edu.cn
作者简介:姚响文,女(汉族),硕士研究生在读.E-mail:852701912@163.com
基金资助:
国家自然科学基金(81271798);国家973项目(2013CB531602)

Construction and identification of SPE1-overexpressing strain of Candida albicans

YAO Xiang-wen, ZHAO Liu-ya, YAN Yu, LU Hui, CAO Ying-ying, JIANG Yuan-ying

New Drug R&D Center, School of Pharmacy, Second Military Medical University, Shanghai 200433, China

Received:2013-05-27 Online:2013-08-28 Published:2013-08-28

摘要/Abstract

摘要： 目的构建白念珠菌SPE1基因高表达菌株。方法将白念珠菌SPE1基因的ORF置于高表达质粒载体pCaEXP的MET3启动子后面,构建pCaEXP-SPE1的高表达质粒,然后采用醋酸锂转染法将高表达质粒转染白念珠菌RM1000中,在SD-ura^-met^-cys^-选择性固体培养基上筛选阳性克隆,抽取基因组进行PCR验证,将验证为阳性转染子的菌落采用Real Time RT-PCR方法进行SPE1基因转录水平的表达验证。结果通过酶切鉴定pCaEXP-SPE1高表达质粒构建正确;通过PCR验证表明SPE1基因整合到亲本菌中的RP10位点;通过Real Time RT-PCR方法筛选出SPE1基因在转录水平高表达的菌株。结论利用高表达质粒载体pCaEXP通过基因同源重组等方法正确构建SPE1基因高表达的白念珠菌。

关键词: 白念珠菌, SPE1, 高表达

Abstract: Objective To construct the overexpression of SPE1 in Candida albicans.Methods We inserted SPE1 open reading frame (ORF) under the control of the MET3 promoter in pCaEXP plasmid to construct pCaEXP-SPE1 plasmids which can overexpress SPE1 gene. Then we use lithium acetate method to transform the pCaEXP-SPE1 into Candida albicans RM1000 and select positive colony in SD-ura^-met^-cys^-selective solid culture medium. PCR was used to investigate the integration and the level of mRNA of SPE1 gene was detected by Real Time RT-PCR.Results SPE1-overexprssing plasmid was exactly established by restriction enzyme digestion.SPE1-overexpressing strain was successfully constructed as confirmed by PCR. And we used Real Time RT-PCR to successfully select a SPE1-overexpressing strain.Conclusions A strain highly expressing SPE1 gene can be successfully constructed by using the pCaEXP plasmid and the gene homologous recombination.

Key words: Candida albicans, SPE1, overexpression

中图分类号:

R379.4

姚响文, 赵柳娅, 颜钰, 鹿辉, 曹颖瑛, 姜远英. 白念珠菌SPE1基因高表达菌株构建及鉴定[J]. 中国真菌学杂志, 2013, 8(4): 198-201.

YAO Xiang-wen, ZHAO Liu-ya, YAN Yu, LU Hui, CAO Ying-ying, JIANG Yuan-ying. Construction and identification of SPE1-overexpressing strain of Candida albicans[J]. Chinese Journal of Mycology, 2013, 8(4): 198-201.

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