Construction of Multi-ribozyme Expression System and Its Characterization of Cleavage on the MDR1/MRP1 Double Target Substrate in vitro

作     者：TIAN Sheng-li ZHENG Suo LIU Shi-de ZHANG Jian-hua XU Dong-ping OHNUMA Takao 

作者机构：Shenzhen Key Laboratory of Microbiology and Gene Engineering College of Life Sciences Shenzhen University Shenzhen 518060 P. R. China Department of Bioengineering 302 Hospital of Beijing Beijing 100039 P. R. China Department of Hematology and Oncology Mount Sinai School of Medicine New York 10029 USA 

出 版 物：《Chemical Research in Chinese Universities》 (高等学校化学研究（英文版）)

年 卷 期：2009年第25卷第2期

页      面：203-210页

核心收录：

学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 081704[工学-应用化学] 07[理学] 08[工学] 0817[工学-化学工程与技术] 09[农学] 071007[理学-遗传学] 0901[农学-作物学] 0836[工学-生物工程] 090102[农学-作物遗传育种] 

基　　金：Supported by Fund of Shenzhen Bureau of Science and Technology  China(No.20008) 

主　　题：Multidrug resistance(MDR) Multidrug resistance-associated protein(MRP1) Multi-ribozyme expression system RNA substrate 

摘      要：To improve catalytic activity of ribozyme on its substrate, the multi-ribozyme expression system was designed and constructed from 20 cis-acting hammerhead ribozymes undergoing self-cleavage with 10 trans-acting hammerhead ribozymes inserted alternatively regularly and the plasmid of pGEM-MDR1/MRP1 used to transcribe the MDR1/MRPl（196/210） substrate containing double target sites was also constructed by DNA recombination. Endonuclease digestion analysis and DNA sequencing indicate all the recombinant plasmids were correct. The clea- vage activities were evaluated for the multi-ribozyme expression system on the MDR1/MRP1 substrate in the cell free system. The results demonstrate that the cis-acting hammerhead ribozymes in the multi-ribozyme expression system were able to cleave themselves and the 72 nt of 196Rz and the 71 nt of 210Rz trans-acting hammerhead ribozymes were liberated effectively, and the trans-acting hammerhead ribozymes released were able to act on the MDR1/MRP1 double target RNA substrate and cleave the target RNA at specific sites effectively. The multi- ribozyme expression system of the [Coat＇A196Rz/Coat＇B210Rz]5 is more significantly superior to that of the [Coat＇A 196Rz/Coat＇B210Rz] 1 in cleavage of RNA substrate. The fractions cleaved by [Coat＇A 196Rz/Coat＇B210Rz]5 on the MDR1/MRP1 substrate for 8 h at observed temperatures showed no marked difference. The studies of Mg^2＋ on cleavage efficiency indicate that cleavage reaction is dependent on Mg^2＋ ions concentration. The plot of lg（kobs） vs. lgc（Mg^2＋） displays a linear relationship between 2.5 mmol/L and 20 mmol/L Mg^2＋. It suggests that Mg^2＋ ions play a crucial role in multi-ribozyme cleavage on the substrate.