论文部分内容阅读
目的 研究针对丙型肝炎病毒 (HCV)特异性锤头样核酶 (Rz)的体外转录及切割活性。方法 设计 3种锤头样核酶 :Rz1、Rz2 分别作用于HCVRNA 5′ 非编码区 (5′ NCR)上核酸序列 136～16 0、313～ 337,Rz3 作用于核心 (C)区上核酸序列 373～ 388。为区别于反义核酸的封闭作用 ,设计在Rz3 的催化环上存在点变异 (G取代A)的变异核酶Rzm用作对照。体外转录出靶HCVRNA和核酶RNA ,在一定条件下 ,将3 2 P标记的靶HCVRNA与核酶RNA按不同浓度比例进行切割反应 ,电泳后放射自显影 ,通过同位素扫描成像分析仪分析来评价核酶切割效率。结果 除Rzm外 ,在生理温度下 ,3种核酶均有活性 ,并随核酶浓度增加而提高 ;核酶切割靶位点距HCV起始密码子近切割效率高。结论 设计并观察到体外具有HCV特异性切割活性的锤头样核酶 ,为进行核酶的细胞内应用研究奠定基础
Objective To study the in vitro transcription and cleavage activity against hepatitis C virus (HCV) specific hammerhead ribozyme (Rz). Methods Three hammerhead-like ribozymes were designed: Rz1 and Rz2 act on the nucleotide sequence of 136-16 0,313-337 in the 5 ’non-coding region of HCV RNA (5’ NCR), and Rz3 act on the nucleic acid sequence in the core region 373 ~ 388. To distinguish from the blocking effect of antisense nucleic acids, a mutant ribozyme Rzm, which had a point mutation (G substitution A) on the catalytic ring of Rz3, was designed and used as a control. In vitro transcription of the target RNA and ribozyme RNA, under certain conditions, the 3 2 P-labeled target HCV RNA and ribozyme RNA at different concentrations in the proportion of cleavage reaction, autoradiography after electrophoresis, by isotope scanning imaging analyzer analysis to evaluate Ribozyme cutting efficiency. Results Except for Rzm, all three ribozymes were active at physiological temperature and increased with the increase of ribozyme concentration. The cleavage target site of ribozyme was near the cutting start codon of HCV efficiently. Conclusions A hammerhead-like ribozyme with HCV-specific cleavage activity was designed and observed in vitro to lay the foundation for the intracellular application of ribozymes