白念珠菌SPE1基因高表达菌株构建及鉴定

目的构建白念珠菌SPEl基因高表达菌株。方法将白念珠菌．SPEl基因的ORF置于高表达质粒载体pCaE—xP的MErF3启动子后面，构建pCaEXP—SPEJ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RMl000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RealTimeRT．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—SPEl高表达质粒构建正确；通过PCR验证表明SPEl基因整合到亲本菌中的RPl0位点；通过RealTimeRT—PCR方法筛选出sPEJ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SPEl基因高表达的白念珠菌。

Construction and identification of SPEl-overexpressing strain of Candida albicans

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.