乙型肝炎病毒特异性核酶与丁型肝炎病毒重组体的构建

Construction of recombinant HDV carring HBV-specific hammerhead ribozyme

目的将重组丁型肝炎病毒基因组作为乙型肝炎病毒特异性核酶的载体，探讨解决核酶在实际应用中的保护性运载、靶向性和特异性问题。方法设计了乙型肝炎病毒特异性锤头型核酶结构，采用重组引物，结合“不对称ＰＣＲ”和“ＭｅｇａＰｒｉｍｅｒＰＣＲ”方法将所设计的锤头型核酶基因替代插入到了型肝炎病毒基因组５'端高变区，重组体经ＰＣＲ初步鉴定后，克隆到Ｔ载体（ｐＴＡｄｖ）Ｔ７启动子下游，经限制性内切酶酶切、序列测定分析。结果与结论证实该重组作为预期的乙型肝炎病毒特异性核酶－丁型肝炎病毒基因组重组体，为进一步的体外切割和细胞内与乙型肝炎病毒相互作用的研究提供了基础。

Hepatitis B virus (HBV) is a DNA virus which replicates through reverse transcription of a RNA intermediate (pregenomeRNA).A recombinant hepatitis D virus (HDV) cDNA with HBV specific hammerhead ribozyme was constructed by a modified PCRderivated tyom asymmetric PCR and Mega primer PCR with primers containing mutation. PCR product was cloned in T-vector underthe control of T7 promotor, and the clone was selected by restrict endonuclease analysis and DNA sequencing. This study suggestthat the recombinant HDV cDNA with HBV specific hammerhead riboryme might be used as a tool for the further cleavage study ofHBC in vitro and in vivo.