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目的以增强绿色荧光蛋白(EGFP)作为报告分子,探讨10-23 DNAzyme对乙型肝炎病毒(HBV)X基因表达的抑制作用。方法设计并合成3种能针对HBV X基因的特异性10-23 DNAzymes,分别命名为DrzHBVX-7、DrzHBVX-8和DrzHBVX-9,能分别特异地识别HBV X基因开放阅读框(ORF)的A1376UG。将内有HBV X全基因片段(不含终止密码)的融合表达质粒pHBx-EGFP与10-23 DNAzyme共转染AD293细胞作为共转染组,用pHBx-EGFP单独转染AD293细胞作为对照组,分别用荧光显微镜观察并用流式细胞仪检测细胞荧光强度,同时用半定量一步法RT-PCR分析融合荧光蛋白HBx-EGFP mRNA的相对表达量。结果各共转染组细胞荧光蛋白HBx-EGFP的表达均明显弱于对照组(P<0．05);各试验组HBx-EGFP mRNA的相对表达量与对照组比较也均明显减少(P<0．05);各共转染组细胞之间HBx-EGFP的表达及HBx-EGFP mRNA的相对表达量差异无统计学意义(P>0．05)。结论10-23 DNAzyme对HBV X基因的表达有特异抑制作用,在HBV感染的基因治疗中会有潜在的应用价值。
Objective To investigate the inhibitory effect of 10-23 DNAzyme on the gene expression of hepatitis B virus (HBV) by using enhanced green fluorescent protein (EGFP) as a reporter. Methods Three specific 10-23 DNAzymes targeting to HBV X gene were designed and synthesized and named as DrzHBVX-7, DrzHBVX-8 and DrzHBVX-9, respectively, which can recognize A1376UG of HBV X gene open reading frame (ORF) . AD293 cells co-transfected with pHBx-EGFP and 10-23 DNAzyme containing full-length HBV X gene (without stop codon) as a co-transfection group and AD293 cells transfected with pHBx-EGFP alone as a control group, Fluorescence microscopy was used to observe the fluorescence intensity of the cells by flow cytometry, and semi-quantitative one-step RT-PCR analysis of fusion protein HBx-EGFP mRNA relative expression. Results The expression of HBx-EGFP in each co-transfection group was significantly weaker than that in the control group (P <0.05). The relative expression of HBx-EGFP mRNA in each co-transfected group was also significantly decreased compared with the control group (P < 0.05). There was no significant difference in the expression of HBx-EGFP and the relative expression of HBx-EGFP mRNA among the co-transfected cells (P> 0.05). Conclusion 10-23 DNAzyme has specific inhibitory effect on the expression of HBV X gene, which may have potential value in the gene therapy of HBV infection.