【摘要】 为鉴定结核分枝杆菌异柠檬酸裂合梅(ICL)特异性的10-23DRz在无细胞体系切割ICLmRNA的活性,并探讨其在不同条件下以及联合应用时对靶mRNA的切割特点,采用计算机软件模拟ICL mRNA的二级结构,据此选择适合的待切割靶点并设计针对相应靶点的特异性10-23DRz(DZ1~DZ5).PCR法扩增获得icl基因并克隆入质粒pET32a+.采用T7 RNA聚合酶体外转录法获取ICL全长mRNA后分别用DZ1~DZ5在无细胞体系中对ICL mRNA进行切割,切割产物经变性聚丙烯酰胺凝胺电泳后用银染法鉴定各DRzs的活性.选择切割活性最强的DZ4考察不同10-23DRz剂量、不同反应时间、不同镁离子浓度条件下及不同错配或突变10-23DRz的切割特点.联合应用DZ1、DZ4及DZ5在无细胞体系中对ICL mRNA进行切割,检测10-23DRz联合应用对切割效率的影响.结果表明,DZ1、DZ3、DZ4及DZ5可在无细胞体系中有效地切割ICL mRNA,其切割效率在30.8%~64.5%之间.对DZ4切割活性的检测发现,其对靶mRNA的切割具有剂量和时间的依赖性;在2~20μmol/L范围内,DZ4的切割活性与Mg2+浓度呈正相关;DZ4单侧底物结合臂上含一个不与靶mRNA配对的碱基时其切割效率大大降低,两侧底物结合臂上各含一个不配对的碱基或活性中心域第6位出现碱基突变(G→C)时,DZ4完全丧失切割活性.联合应用2种或2种以上10-23DRz可显著增强对底物RNA的切割效率.10-23 DRz特异、有效地切割结核分枝杆菌ICL全长mRNA并显示一定的叠加效应,有望用于抗结核分枝杆菌潜伏感染的基因治疗.更多还原

【Abstract】 To evaluate the cleavage activity of 10-23 deoxyribozyme（10-23DRz） targeting Mycobacterium tuberculosis isocitrate lyase（ICL） mRNA,and to investigate the cleavage characteristics of 10-23DRzs under various conditions,the cleavage sites along the ICL mRNA were determined according to the predicted secondary structure of ICL mRNA by RNA STRUCTURE 3.7 program.Five 10-23 DRz（DZ1～DZ5） were designed according to the cleavage sites and 10-23 model.icl gene was amplified by PCR and cloned into plasmid pET32a+.The full length ICL mRNA was obtained in vitro transcription using T7 RNA polymerase.The cleavage reaction of DZ1～DZ5 was performed in cell-free system and detected by denature polyacrylamid gel electrophoresis followed by silver staining.The specificity and cleavage characteristics at various dosage of 10-23 DRz,time and concentration of Mg²⁺ of DZ4 were also studied in cell-free system.The combinatorial cleavage activity was studied using DZ1,DZ4 and DZ5 in combination.Four of the five designed 10-23DRz（DZ1,DZ3,DZ4 and DZ5） could cleavage target mRNA specifically.The cleavage efficiency of these four 10-23DRzs to ICL mRNA was from 30.8% to 64.5%.The cleavage activity of DZ4 was dosage and time dependent,and was Mg²⁺ concentration dependent when the concentration of Mg²⁺ was within the scope of 2～20 μmol/L.The cleavage activity of DZ4 was dramatically dropped when a mismatch appeared in one of the substrate-binding regions.When a mismatch in each of the substrate-binding regions or a mutation occurred in the catalytic core,DZ4 lost cleavage activity completely.The cleavage activity of combination of 10-23DRz was higher than each 10-23 DRz alone.10-23 DRz can specifically cleavage ICL mRNA of M.tuberculosis with high efficiency and show a combinatorial effect.更多还原