丁型肝炎病毒核酶反式切割HBV mRNA片段的实验研究  

Experimental study on HDV ribozyme in vitro cleaving the HBV derived RNA fragment

作　　者：潘光锦[1] 韩金祥[1] 

机构地区：[1]山东省医药生物技术研究中心卫生部生物技术药物重点实验室,济南250062

出　　处：《中华实验和临床病毒学杂志》2003年第2期149-152,共4页Chinese Journal of Experimental and Clinical Virology

基　　金：山东省自然基金资助 (Q990 15 )

摘　　要：目的　探讨反式作用丁型肝炎病毒 (HDV)核酶体外切割乙型肝炎病毒 (HBV)mRNA片段的可行性。方法　将化学合成的核酶cDNA克隆到含有T7启动子的载体PGEM 4Z中。利用体外转录技术转录出核酶及底物 ,研究其体外切割活性。利用E H作图法进行核酶的酶促动力学研究。结果　在体外实验中显示两酶均能成功的将底物切割 ,37℃温浴 90min的切割百分率为 5 0 %和5 1%。利用E H作图法进行的酶促动力学研究中求得Rc1、Rc2的Km值分别为 0 6 1μmol L、0 5 8μmol L,Kcat值分别为 :0 6 4·min 1 、0 6 0·min 1 。结论　反式作用HDV核酶对非HDV底物 HBVmRNA片段的成功切割为寻找新的HBV的反义抑制手段开辟了途径。Objective To explore the possibility of transacting hepatitis D virus (HDV) ribozyme cleaving in vitro the hepatitis B virus (HBV) mRNA fragments Methords According to the established pseudoknot like structure, its' H1 domain was changed to design the transacting HDV ribozyme Rc1 and Rc2, which targeted the 701 713 site and 776 788 site of HBV C domain After the chemically synthesised cDNA of the ribozyme was cloned into the vector PGEM 4Z, the transacting HDV ribozyme was transcriped using in vitro transcription technology The in vitro cleavage characteristics of the ribozyme were studied and the kinetic parameters (Kcat and Km) were determined by Eadie Hofstee plotting Results Both the two ribozymes had the ability to cleave the substrate, the cleavage percentage at 37℃ for 90 minutes were 50% and 51% According to the Eadie Hofstee plot, the Km of the Rc1 and Rc2 were 0 61 μmol and 0 58 μmol, the Kcat were 0 64·min 1 and 0 60·min 1 ,respectively Conclusion The cleaving ability of trans acting HDV ribozyme on non HDV RNA fragment was tested The resuls showed a new potential of the antisense antisense regent for HBV gene therapy

关 键 词：丁型肝炎病毒 核酶 HBV mRNA片段 实验 乙型肝炎病毒 体外切割 

分 类 号：R346[医药卫生—基础医学]