丙型肝炎病毒特异性锤头样核酶体外转录及切割活性的初步研究

Study of transcription and cleavage in vitro of HCV with HCV specific hammerhead ribozyme

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 。

Objective To study the effect of specific hammerhead ribozyme (Rz) to hepatitis C virus (HCV) in vitro. Methods Rz 1 Rz 2 were designed to cleavage at 5′-NCR nucleotide positions under 136～160 and 313～337, Rz 3 was designed to cleavage at C region nucleotide position under 373～388. As a control, cleavage deficient Rzm that have A→G point mutations in the catalytic loop of the hammerhead domain. 32P-labeled transcript of target HCV RNA was incubated with gel-purified Rz ( Rz 1, Rz 2, Rz 3 and Rzm ) respectively at different concentration based on specified condition and autoradiographed after denaturing gel-electrophoresis. Results Except Rzm, Rz 1 Rz 2 Rz 3 were active at 37 ℃ and more so at higher concentration, and more so with cleavage site nearly to the HCV initial code. Conclusions The HCV specific hammerhead ribozyme can be designed in vitro, further study about cleavage in vitro and in vivo will continue.