核酶在细胞外切割HCV核心区RNA的研究  

Extracellular cleavage of viral RNA by hepatitis C virus core gene RNA specific hammerhead ribozyme

作　　者：丁芹[1] 龚国忠[1] 郑宣鹤[1] 游学科[2] 

机构地区：[1]湖南医科大学附属第二医院肝病研究中心 [2]湖南医科大学附属第二医院医学实验中心分子生物室

出　　处：《中华微生物学和免疫学杂志》2001年第3期318-320,共3页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目!(3 9670 67)

摘　　要：目的　研究核酶在细胞外切割HCV的作用。方法　设计核酶cDNA序列。构建核酶重组质粒及HCV核心区基因cDNA重组质粒。分别进行体外转录 ,并加入γ 3 2 PATP以标记HCVRNA。将核酶RNA、HCV核心区RNA、RNA酶抑制剂及反应缓冲液混合温育 ,以核酶RNA切割HCVRNA。通过变性聚丙烯酰胺凝胶电泳及放射自显影来分析结果。结果　β 半乳糖苷酶表型筛选均可见蓝色及白色菌落生长 ;核酶重组质粒直接测序结果见预期核苷酸序列 ;HCV重组质粒限制性酶切分析见 410bp条带。核酶切割反应显示 :反应时间为 15和 30min时可见 45 3、30 7、146nt3条带 ;反应时间为 6 0min时仅见 30 7及 146nt2条带。结论　核酶重组质粒构建成功 ,所设计核酶对HCV有切割作用。Objective To design and synthesize the cDNA fragment of hammerhead ribozyme, which was hepatitis C virus core gene RNA-specific, and investigate its extracellular cleavage of hepatitis C virus core gene RNA. Methods Design the ribozyme cDNA fragment and construct recombinants. Clone HCV core gene cDNA to pGEM-7Zf (+) plasmids. Get ribozyme RNA and HCV RNA through in vitro transcription. Mix ribozyme RNA, HCV RNA, ribozyme reaction buffer and RNasin in certain ratio. Incubate them at 37℃. Analyze the result by polyacrylamide gel electrophoresis and auto photographing. Result Both white and blue bacteria groups could be seen in the white and blue scanning system. Sequencing showed the sequence from 130 to 82 was combinative to ribozyme cDNA. As to HCV core gene recombinant, after EcoRⅠ and Hind Ⅲ cleavage, a band could be seen at about 410bp on agarose gel electrophoresis. Auto-photographing showed HCV RNA had only one band at 453nt. After ribozyme reaction, however, three bands could be seen when the incubation time was 15 minutes or 30 minutes, which were 453nt, 307nt, 146nt, while only two bands were seen at 307nt and 146nt when the incubation time was 60 minutes. Conclusion The result showed that the HCV core gene RNA-specific hammerhead ribozyme expression vector had been successfully constructed, and the ribozyme could cleave HCV RNA.

关 键 词：核酶 丙型肝炎病毒 基因治疗 RNA 丙型肝炎 

分 类 号：R512.62[医药卫生—内科学]