摘要
目的构建白念珠菌SPE 1基因高表达菌株。方法将白念珠菌SPE 1基因的ORF置于高表达质粒载体pCaEXP的MET3启动子后面,构建pCaEXP-SPE 1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SPE 1基因转录水平的表达验证。结果通过酶切鉴定pCaEXP-SPE 1高表达质粒构建正确;通过PCR验证表明SPE 1基因整合到亲本菌中的RP10位点;通过Real Time RT-PCR方法筛选出SPE 1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SPE 1基因高表达的白念珠菌。
Objective To construct the overexpression of SPE 1 in Candida albicans. Methods We inserted SPE 1 open read- ing frame （ORF） under the control of the MET3 promoter in pCaEXP plasmid to construct pCaEXP- SPE 1 plasmids which can over- express SPE 1 gene. Then we use lithium acetate method to transform the pCaEXP- SPE 1 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 SPE 1 gene was detected by Real Time RT-PCR. Results SPE 1-overexprssing plasmid was exactly established by restriction en- zyme digestion. SPE 1-overexpressing strain was successfully constructed as confirmed by PCR. And we used Real Time RT-PCR to successfully select a SPE 1-overexpressing strain. Conclusions A strain highly expressing SPE 1 gene can be successfully construc- ted by using the pCaEXP plasmid and the gene homologous recombination.
出处
《中国真菌学杂志》
CSCD
2013年第4期198-201,共4页
Chinese Journal of Mycology
基金
国家自然科学基金(81271798)
国家973项目(2013CB531602)
关键词
白念珠菌
SPE1
高表达
Candida albicans
SPE 1
overexpression
