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[Preparation and identification of activity of antiplatelet-derived growth factor receptor beta subunit hammerhead
1Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
Objective:
To study the preparation and cleavage activity of ribozyme directed against platelet-derived growth factor (PDGF) receptor beta subunit gene transcript in vitro.
Methods:
PDGF receptor B subunit gene fragments were cloned into T-vector under the control of T7 promotor. 32P-labeled PDGF receptor beta subunit transcripts as target-RNAs were transcribed in vitro and purified by PAGE. Ribozyme gene designed by computer targeting the extracellular domain was cloned into vector P1.5 between 5'-cis ribozyme and 3'-cis ribozyme. 32P-labeled ribozyme transcripts was gel-purified, incubated with target-RNAs at different conditions and autoradiographed after denaturing gel-electrophoresis.
Results:
The preparation of ribozyme was correct. It was active at 37 degrees C. The optimal temperature was 50 degrees C, Km=13,20 nm, Kcat=0.28 min-1. The cleavage efficiency was up to 78.3%.
Conclusions:
Ribozyme prepared in vitro possesses specific catalytic activity.
