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ICL mRNA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法扩增获得纠基因并克隆入质粒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Mg^2＋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．10-23DRz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．
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 Mg^2＋ of DZA 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 Mg^2＋ concentration dependent when the concentration of Mg^2＋ was within the scope of 2 - 20μmol/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. tubewulosis with high efficiency and show a combinatorial effect.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2006年第2期151-157,共7页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金资助项目(No.30270584)~~
