【摘要】 目的:探讨10-23 DNA enzym e对乙型肝炎病毒C基因体外转录产物的特异切割活性。方法:设计并合成3种能针对乙型肝炎病毒C基因开放阅读框A1816UG的特异性脱氧核酶,分别命名为D rzBC-7、D rzBC-8和D rzBC-9。应用体外转录的方法获得相应的底物RNA,观察D rzBC-7、D rzBC-8和D rzBC-9对其的体外切割效应。以D rzBC-9为例,观察不同浓度的M gC l2对体外切割效率的影响,根据L inew eaver Burk作图法,计算相关的酶动力学参数Km、K cat和K cat/Km。结果:通过体外转录获得用于切割反应的底物RNA,其大小为300 nt。在特定的切割条件下,D rzBC-7、D rzBC-8和D rzBC-9均能对相应的底物RNA进行有效的切割,切割产物分别为109 nt和191 n。t以D rzBC-9为例,在切割反应体系中缺乏M gC l2时,未见有切割反应。M gC l2浓度在150 mm o.lL-1时达到最高切割效率,再提高M gC l2的浓度,切割效率未见明显增大。酶动力学参数Km、K cat和K cat/Km分别为1.4×10-9m o.lL-1,1.6 m in-1和1.1×109m o.lL-1.m in-1。结论:针对乙型肝炎病毒C基因的10-23 DNA enzym e在体外对相应的转录产物有特异切割作用。更多还原

【Abstract】 Objective: To investigate the cleavage activities of 10-23 DNA enzymes targeting at HBV C gene mRNA in vitro.Methods: 10-23 DNA enzymes named DrzBC-7,DrzBC-8 and DrzBC-9 specific to HBV C gene ORF A¹⁸¹⁶UG were designed and synthesized.HBV C gene mRNA was obtained by in vitro transcription method.Cleavage activities were observed in vitro.The influence of MgCl₂ concentration on RNA cleaving activity was examined with DrzBC-9.Values of kinetic parameters including Km,Kcat and Kcat/Km were calculated accordingly.Results: Targeted substrate mRNA with the size of 300 nt was obtained by transcription in vitro.Under the certain cleavage conditions,DrzBC-7,DrzBC-8 and DrzBC-9 all efficiently cleaved target mRNA at specific sites in vitro.Cleavage products of 109 nt and 191 nt were obtained.No cleavage occurred without MgCl₂.The most efficient cleavage was obtained at 150 mmol·L^-1 MgCl₂.The efficiency of cleavage did not increase when the MgCl₂ concentration was more than 200 mmol·L^-1.The kinetic parameters,Km,Kcat and Kcat/Km for DrzBC-9 were 1.4×10^-9mol·L^-1,1.6 min^-1 and 1.1×10⁹mol·L^-1·min^-1,respectively.Conclusion: 10-23 DNA enzymes targeting at HBV C gene mRNA possess the specific cleavage activities in vitro.更多还原