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目的探讨靶向白色假丝酵母菌分泌型天冬氨酸蛋白酶(SAP)的小分子干扰RNA(siRNA)对白色假丝酵母菌SAP的基因表达影响,以期为RNA干扰技术抑制真菌毒力提供实验基础。方法设计并合成SAP靶向的siRNA,用醋酸锂转染白色假丝酵母菌,试验设空白对照组、错义对照组、SAP siRNA组,采用RT-PCR测定转染后白色假丝酵母菌SAP mRNA表达水平,制备BSA培养基测定天冬氨酸蛋白酶活性及小鼠毒力实验。结果与空白对照组、错义对照组相比,转染48h后,siRNA组白色假丝酵母菌中SAP mRNA表达明显下调为(0.51±0.05),siRNA组天冬氨酸蛋白酶活性PA值为(0.84±0.04)均高于空白及错义对照组,小鼠30d死亡率显著降低为20.0%。结论化学合成的靶向SAP siRNA能够下调白色假丝酵母菌中SAP基因的表达,降低白色假丝酵母菌的毒力,该实验为进一步研究应用RNA干扰技术抗真菌治疗奠定基础。
Objective To investigate the effect of small interfering RNA (siRNA) targeting Candida albicans secreted aspartic proteinase (SAP) on the gene expression of Candida albicans SAP in order to provide experimental evidence for the inhibition of fungal virulence by RNA interference basis. Methods siRNA targeting SAP was designed and synthesized. Candida albicans was transfected with lithium acetate. The blank control group, the missense control group and the SAP siRNA group were designed and transfected with Candida albicans SAP mRNA expression levels were prepared BSA medium determination of aspartic protease activity and mouse virulence experiments. Results Compared with the blank control group and the missense control group, the mRNA expression of SAP in Candida albicans was significantly down-regulated to (0.51 ± 0.05) and the asparaginase activity in siRNA group was ( 0.84 ± 0.04) were higher than the blank and missense control group, the mice 30d mortality was significantly reduced to 20.0%. CONCLUSIONS: Chemically synthesized targeted siRNAs can down-regulate the expression of SAP gene in Candida albicans and reduce the virulence of Candida albicans, which lays a foundation for further research on anti-fungal therapy using RNA interference technology.