Clone, expression and cleavage activity of anti-caspase-7 hammerhead ribozyme in vitro / 中华肝脏病杂志

ABSTRACT

<p><b>OBJECTIVE</b>To design hammerhead ribozymes against mouse caspase-7 and to study their expression and cleavage activity in vitro.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>Two 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.</p><p><b>CONCLUSIONS</b>Rz333 can site-specifically cleave caspase-7 mRNA.</p>