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目的构建白色念珠菌CaTIP41基因高表达菌株,并观察其高表达对细胞耐受遗传毒性试剂MMS的影响。方法将白色念珠菌CaTIP41基因的ORF置于高表达质粒载体pCaEXP的MET3启动子后,构建pCaEXP-CaTIP41高表达质粒,线性化后整合到基因组的RP10位点,利用半定量PCR和RT-PCR方法检测CaTIP41基因转录水平,利用平板表型试验结合菌株存活率试验检测CaTIP41基因高表达对细胞耐受遗传毒性试剂的影响。结果成功构建了高表达载体pCaEXP-CaTIP41;线性化后的载体成功整合到基因组上RP10位点,其中4号转化子中CaTIP41基因转录水平较高,为野生型菌株的4.23倍;在MMS胁迫条件下,CaTIP41基因高表达菌株存活率明显下降。结论利用pCaEXP载体成功构建了CaTIP41基因高表达菌株,且CaTIP41基因高表达会抑制细胞对遗传毒性试剂MMS的耐受。
Objective To construct a highly expressed CaTIP41 gene of Candida albicans and observe its effect on the tolerance of genotoxic agents to MMS. Methods The ORF of Candida albicans CaTIP41 gene was placed in the MET3 promoter of high expression plasmid vector pCaEXP to construct the high expression plasmid pCaEXP-CaTIP41. After linearization, the ORF of CaTIP41 gene was integrated into RP10 locus. Using semi-quantitative PCR and RT-PCR The transcription level of CaTIP41 gene was detected and the effect of CaTIP41 gene overexpression on the cytotoxicity of genotoxic agents was tested by plate phenotype assay and strain survival test. Results The vector pCaEXP-CaTIP41 was successfully constructed. The linearized vector was successfully integrated into the genome of RP10. The transcription level of CaTIP41 gene in transformant No.4 was 4.23 times that of the wild-type strain. Under the conditions of MMS stress , The viability of CaTIP41 highly expressed strains was significantly decreased. Conclusion The CaTp41 gene highly expressed strain was successfully constructed by using pCaEXP vector and the high expression of CaTIP41 gene inhibited the tolerance of the cells to the genotoxic agent MMS.