乙型肝炎病毒特异性核酶与丁型肝炎病毒重组体的体外转录及切割活性

Study of transcription and cleavage in vitro of HDV with HBV-specific hammerhead ribozyme

研究乙型肝炎病毒（ＨＢＶ）特异性核酶（简称ｒＲＺ）体外转录及切割ＨＢＶ的活性。方法将８３１ｂｐ的ＨＢＶＣ基因片段克隆于Ｔ载体Ｔ７启动子下游，３２Ｐ标记转录后作为靶ＲＮＡ（３２Ｐ－ｒＨＢＶ）。ｒＲＺ、ｒＨＤＶＲＺＡ插入有核酶结构的丁型肝炎病毒基因组重组体）和ｒＨＤＶＲＺＰ（部分ＨＤＶ基因组重组体）进行非标记的大量转录，转录产物经凝胶纯化后，与３２Ｐ－ｒＨＢＶ按一定比例和条件保温切割，电泳后放射自显影。结果ｒＲＺ，ｒＨＤＶＲＺＰ，ｒＨＤＶＲＺＡ在３７Ｃ有活性，并随温度升高而提高，体外观察到重组体的切割活性，证明所设计的核酶结构是正确的。结论将核酶基因插人到ＨＤＶ基因组中，使其具有特异性切割ＨＢＶ的活性，可实现核酶的保护性和靶向性运载，为下一步重组体的体内应用研究奠定了基础。

Objective To study the effect of hepatitis B virus(HBV) specific ribozyme(RZ) andrecombinant hepatitis D virus(HDV) inserting hammerhead ribozyme(rHDVRZP and rHDVRZA). Methods831 bp HBV C gene fragment was cloned under the control of T7 promoter, 32P-labeld HBV transcriptwas incubated with gel-purified RZ, rHDVRZA, rHDVRZP at different temperature and auter after denaturing gel-electrophoresis. Result These results show that rRZ, rHDVRZA, rHDVRZP wereactive at 37 and more so at higher themperatures. Conclusion Recombinant Deft virus could serve as avector for the delivery of a ribozyme speciific for hepatitis B virus cleavage. Our data demonstrate thevalue of recombinant ribozyme as potential therapeutic agents for treatment of HBV infection. Furtherstudy about cleavage in vitro and in vivo will continue.