[Preparation and identification of activity of antiplatelet-derived growth factor receptor beta subunit hammerhead ribozyme in vitro].
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.
Lu
C
C
Department of Gastroenterology, Changzheng Hospital, Second Military Medical University, Shanghai 200003, China.
Chen
Y
Y
Zhang
Z
Z
Wen
L
L
Guo
Y
Y
Zhang
X
X
Jin
Y
Y
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002478:Cells, Cultured; D051379:Mice; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
D016337:RNA, Catalytic; D012333:RNA, Messenger; D020797:Receptor, Platelet-Derived Growth Factor beta