HBV特异性核酶与HDV重组体的体外转录及切割活性研究

Study of transcription and deavage in vitro of HDV with HBV-speciffc hammerhead ribozyme

目的利用基因重组技术,用核酶基因置换HDV部分基因,分别用HBV特异性核酶(rRZ)和核酶-HDV重组体(rHDVRZA、rHDVRZB和rHDVRZC)研究体外转录与剪切HBV的活性。方法 将含有83lbp的HBV C基因片段的质粒pTA-HBV用BamHI消化成线性后,体外转录获取5’端32P标记靶HBV C RNA。将获得的3个核酶-HBV重组体rHDVRZA、rHDVRZB和rHDVRZC克隆于pGEM-T载体T7启动子下游进行非标记的大量转录。分别将rRZ、rHDVRZA、rHDVRZB和rHDVRZC与卫32P-HBV C RNA按一定比例和条件保温切割。结果 体外实验证明rRZ、rHDVRZA、rHDVRZB和rHDVRZC在37℃具有酶切活性。提示所设计的核酶-HDV重组体rHDVRZA和rHDVRZB中核酶结构正确。结论在HDV基因组不同位置插入核酶,能够维持酶正确结构的核酶-HDV具有体外特异性切割HBV的活性。

Objective To study the effect of hepatitis B virus (HBV) cleavage by HBV specific ribozyme (RZ) and recombi-nant hepatitis D virus (rHDV) harbouring hammerhead RZ(rHDVRZA,rHDVRZB and rHDVRZC) .Methods The pTA-HBV vector including 831bp HBV C gene fragment was digested with BamHI and cloned under the control of T7 promoter, and 32P labeled HBV C transcript was incubated with gel-purified rRZ,rHDVRZA,rHDVRZB and rHDVRZC at different temperature and autoradiographed after denaturing gel-electrophoresis.Results Our results showed that rRZ,rHDVRZA,rHDVRZB and RHDVRZC were active at 37℃. Conclusion rHDV could serve as a vector for the delivery of the RZ specific for HBV cleavage. Our data demonstrated the value of rRZ as potential therapeutic agents for HBV infection.