抗DEN-2基因核酶的克隆及其体外切割活性的研究

In vitro cleavage of dengue virus RNA by a hammerhead ribozyme

目的 探索核酶抗登革病毒活性。方法 根据DEN 2基因结构的特点 ,按照锤头状核酶的结构模型和作用模式 ,设计、合成针对 172～ 174GUC位点的核酶基因 ;定向插入质粒pGEM 3Zf(+)的SacⅠ和SalⅠ位点之间 ;核酶基因克隆和DEN 2靶基因克隆分别进行体外转录 ,生成核酶和靶RNA ,体外进行切割反应。结果 核酶与登革病毒靶序列体外产物电泳见预期切割条带。结论 该核酶具有切割相应靶RNA的活性 。

Objective To explore the cleavage activity of hammerhead ribozyme to dengue virus. Methods A hammerhead ribozyme specific to 172-174 GUC triplet of dengue virus 2 gene was cloned into the SacⅠ and SalⅠ site of vector pGEM3Zf(+). The recombinant plasmid was screened out by sequence analysis. The hammerhead ribozyme and its target RNA were transcribed and then mixed up and incubated in vitro. The cleavage product was analyzed by PAGE and silver-staining. Results The target RNA fragment was cleaved by the hammerhead ribozyme. Conclusion Hammerhead ribozyme possesses the cleavage activity to dengue virus and can be used in antiviral studies in vitro.