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目的 探讨用丁型肝炎病毒 (HDV)基因组来包埋HBV靶向性核酶对核酶体内外活性产生的影响。方法 用和HBV靶基因体外转录产物在不同反应条件下温育对HDV 核酶重组体的体外切割活性定量分析 ;与HBV基因组共转染Huh 7细胞以考察核酶在细胞内对HBV基因表达水平的抑制。结果 体外实验发现 ,温度及核酶和底物的二级结构对包埋于HDV序列的核酶体外切割活性均有较大的影响。提高反应温度或消除二级结构都可使HDV包埋核酶的体外活性达到明显效果 ,与相同条件的裸露核酶活性没有太大差异。而细胞实验则表明 ,活性明显优于裸露核酶 ,可以将靶基因的表达抑制到极低水平。结论 HDVRNA序列对所包埋核酶体外活性有一定的抑制作用 ,在细胞内则使核酶的活性得到显著增强
Objective To investigate the effect of hepatitis B virus (HBV) targeting HBV ribozyme on the in vivo and in vitro activities of ribozymes. Methods Quantitative analysis of in vitro cleavage activity of recombinant HDV ribozyme incubated with HBV transcripts of target gene in vitro under different reaction conditions; cotransfection of Huh 7 cells with HBV genome to investigate the effect of ribozyme on HBV gene expression Of inhibition. Results In vitro experiments showed that the temperature and the secondary structure of ribozyme and substrate had a great influence on the in vitro cleavage activity of the ribozyme embedded in HDV sequence. Enhancing the reaction temperature or eliminating the secondary structure can make HDV embedding ribozyme in vitro achieve a significant effect in vitro, which is not much different from the naked ribozyme activity under the same conditions. The cell experiments showed that the activity was significantly better than the naked ribozyme, the target gene expression can be suppressed to a very low level. Conclusion The HDV RNA sequence can inhibit the in vitro activity of the embedded ribozyme and enhance the activity of ribozyme in the cell