白念珠菌RAS1基因高表达菌株的构建及鉴定

摘要

目的：构建白念珠菌RAS1基因高表达菌株pCaEXP⁃RAS1⁃CAI4。方法：将白念珠菌RAS1基因的开放阅读框（ ORF）置于高表达载体pCaEXP的启动子MET3之后，构建高表达质粒载体pCaEXP⁃RAS1。将整合的重组质粒转化进入大肠杆菌DH5α，经扩增后采用质粒抽提试剂盒大量制备pCaEXP⁃RAS1质粒。单酶切线性化质粒，将线性化的pCaEXP⁃RAS1经醋酸锂法转化入白念珠菌CAI4细胞内，随后在SD⁃ura⁃met⁃cys选择性培养基获得转化子，构建pCaEXP⁃RAS1⁃CAI4菌株。挑取单克隆菌落，经菌落PCR验证阳性转化子，并采用实时定量PCR检验RAS1基因在pCaEXP⁃RAS1⁃CAI4菌株的表达水平。结果：酶切及测序鉴定表明高表达质粒载体pCaEXP⁃RAS1构建成功，实时定量PCR检验转化子RAS1基因在pCaEXP⁃RAS1⁃CAI4菌株的表达水平，表明pCaEXP⁃RAS1⁃CAI4菌株构建成功。结论：通过高表达质粒载体pCaEXP⁃RAS1可以成功构建pCaEXP⁃RAS1⁃CAI4菌株。%Objective:To construct the overexpression RAS1 gene of Candida albicans. Methods:We put the open reading frame of RAS1 gene under the control of MET3 promoter in plasmid pCaEXP to construct recombinant plasmid pCaEXP⁃RAS1 which can overex⁃press RAS1 gene. The plasmid pCaEXP⁃RAS1 was transformed into Escherichia coli DH5α for amplification and extracted by Plasmid Maxi Kit. Then the recombinant plasmid was linearized by cutting of restriction enzyme, and the linear recombinant plasmid was intro⁃duced into Candida albicans CAI4 by lithium acetate to select positive colonies on the plates of SD⁃ura⁃met⁃cys selective culture medi⁃um. Realtime RT⁃PCR was used to detect the expression level of mRNA of RAS1gene. Results:RAS1⁃overexpression plasmid was ex⁃actly established by restriction enzyme digestion. RAS1⁃overexpression strain was constructed as confirmed by quantitative real⁃time PCR. RAS1⁃overexpression strain was selected by quantitative real⁃time PCR. Conclusions: The RAS1⁃overexpression strain can be successfully constructed by recombinant pCaEXP⁃RAS1 plasmid.