Activity identification of anti-caspase-3 mRNA hammerhead ribozyme in both cell-free condition and BRL-3A cells
10.3760/j.issn:0366-6999.2001.06.010
- VernacularTitle:抗Caspase-3核酶的体外及BRL-3A细胞内活性鉴定
- Author:
Renhuan XU
1
;
Jing LIU
;
Xiaqiu ZHOU
;
Qing XIE
;
Hong YU
;
Youxin JIN
;
Dan LIAO
Author Information
1. Ruijin Hospital Shanghai Second Medical University;
- Keywords:
activity identification * ribozyme * BRL-3A * caspase-3 * apoptosis inhibitor
- From:
Chinese Medical Journal
2001;114(6):606-611
- CountryChina
- Language:Chinese
-
Abstract:
Objective To study the transcription effects and cleavage activities of rat caspase-3 specific hammerhead ribozyme (Rz107) in both cell-free conditions and BRL-3A cells. Methods Rat caspase-3 gene fragment was cloned into the pGEM-T EASY vector under the T7 promoter control. The 32 P-labeled caspase-3 transcript was the target-RNA. Rz107 genes designed against caspase-3 mRNA were cloned into vector p1.5 between 5'-cis-Rz and 3'-cis-Rz. 32 P-labeled ribozyme transcripts were incubated with target-RNAs at different conditions and autoradiographed after denaturing gel-electrophoresis. Rz107 was electroporated into BRL-3A cells and the Rz107 expression was analyzed by RT-PCR. Results In cell-free conditions, Rz107 was active at 37℃. The optimal temperature was 50℃. The Km and kcat were 14.13?nmol/L and 2.31*min-1 respectively. Intracellular cleavage efficiency of Rz107 was 37%, as analyzed by RT-PCR. This indicated that the design of Rz107 was correct, and Rz107 had the activity of common enzymes. Conclusions Rz107 in cell-free conditions possessed perfect specific catalytic cleavage activity, and it can also cleave the target RNA successfully in cells. The results illustrate the feasibility of ribozyme therapy as a potential alternative approach for treating liver disease caused by apoptosis.
