丁型肝炎病毒基因组RNA包埋锤头状核酶的活性研究  

Investigation on activities of hammerhead ribozyme embedded in genomic RNA of hepatitis delta virus

作　　者：李晓娟[1] 况二胜[2] 戴炜[1] 杨复华[2] 王敏[1] 王火生[1] 周伯平[1] 

机构地区：[1]深圳市东湖医院,深圳市肝病研究所,518020 [2]武汉大学生命科学学院

出　　处：《中华实验和临床病毒学杂志》2005年第1期12-15,共4页Chinese Journal of Experimental and Clinical Virology

基　　金：广东省自然科学基金资助项目 (0 12 0 2 7)

摘　　要：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 。Objective To develop HDV as a vehicle to deliver hammerhead ribozyme into hepatocytes, the effects of modified HDV was assessed on the activity of embedded hammerhead ribozyme in vitro and in vivo. Methods In vitro activity of ribozyme or HDV-driven ribozyme was assessed by incubating with the [α-^(32)P]-ATP labeled HBV RNA substrates at different temperature. Huh-7 cells were cotransfected with ribozyme or HDV-ribozyme chimera and HBV genome, by which inhibition of ribozymes on HBV transcription in vivo were examined. Results The results indicated that both temperature and secondary structure influenced the cleavage activity of HDV-driven ribozyme significantly. When the factors were eliminated, the HDV-driven ribozyme could act as well as its counterpart naked ribozyme. While in cultured cells the HDV-driven ribozyme had higher inhibition to HBV gene expression than that of ribozyme alone. Conclusion The results demonstrated that HDV may weaken the activity of embedded ribozyme in vitro, but make it enhanced in cultured cells. Thus, this study could provide a useful evidence to develop HDV as vector for liver-special delivery of ribozyme to against chronic HBV infection.

关 键 词：锤头状核酶 丁型肝炎病毒 包埋 基因组RNA 体外切割 细胞内 活性 二级结构 基因表达水平 重组体 

分 类 号：R346[医药卫生—基础医学]