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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 investigate the feasibility of in vitro cleavage of Hepatitis B virus (HBV) mRNA fragment by trans-acting hepatitis D virus (HDV) ribozyme. Methods The chemically synthesized ribozyme cDNA was cloned into the vector PGEM 4Z containing the T7 promoter. In vitro transcription was used to transcribe ribozymes and substrates to study their in vitro cleavage activity. Enzymatic kinetics of ribozyme was studied by using E H mapping. Results In vitro experiments showed that both enzymes successfully cleaved the substrate, and the cleavage rates were 50% and 51% at 37 ℃ for 90 min. The Km values of Rc1 and Rc2 calculated by EH mapping method were 0 6 1μmol L and 0 58μmol L, respectively. The Kcat values were 0 6 4 · min 1 and 0 6 0 · min 1 . Conclusion The successful cleavage of HBV mRNA fragment by non-HDV substrate by trans-acting HDV ribozyme opens up a new way to find new antisense inhibitor of HBV.