Clone, expression and cleavage activity of anti-caspase-7 hammerhead ribozyme in vitro.
- Author:
Wei ZHANG
1
;
Qing XIE
;
Xia-qiu ZHOU
;
Shan JIANG
;
You-xin JIN
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Base Sequence; Caspase 7; Caspase Inhibitors; Caspases; genetics; Cloning, Molecular; Mice; Mice, Inbred BALB C; Molecular Sequence Data; RNA, Catalytic; biosynthesis; genetics; metabolism; RNA, Messenger; biosynthesis; genetics
- From: Chinese Journal of Hepatology 2004;12(11):684-687
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo design hammerhead ribozymes against mouse caspase-7 and to study their expression and cleavage activity in vitro.
METHODSThe secondary structures of ribozyme and caspase-7 genes were analyzed and simulated by computer. Ribozymes DNA sequences were synthesized by automatic synthetic apparatus. Caspase-7 DNA sequence was acquired by reverse transcription PCR. Ribozymes and caspase-7 DNA sequences were separately cloned into pBSKneo U6 and pGEM-T vectors. Ribozymes and caspase-7 mRNA were obtained by transcription in vitro, and ribozymes cleavage activity was identified by cleavage experiment in vitro.
RESULTSTwo ribozymes named Rz333 and Rz394 targeting 333 and 394 sites in caspase-7 mRNA were designed by computer software, and their DNA sequences were synthesized. The expression vector of caspase-7 and plasmids containing Rz333 and Rz394 were reconstructed successfully. Ribozymes and caspase-7 mRNA were expressed by in vitro transcription. In vitro cleavage experiments showed that Rz333 cleaved caspase-7 mRNA and produced 243nt and 744nt segments. The cleavage efficiency is 67.98%, while Rz394 cannot cleave caspase-7 mRNA.
CONCLUSIONSRz333 can site-specifically cleave caspase-7 mRNA.
