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AIM:To design a hammerhead ribozyme targeting humantelomerase reverse transcriptase(hTERT)and clone it’s genefor future use in the study of tumor gene therapy.METHODS:Using the software RNAstructure,the secondarystructure of hTERT mRNA was predicted and the cleavagesite of ribozyme was selected.A hammerhead ribozymetargeting this site was designed and bimolecular fold betweenthe ribozyme and hTERT was predicted.The DNA encodingthe ribozyme was synthesized and cloned into pGEMEX-1and the sequence of the ribozyme gene was confirmed byDNA sequencing.RESULTS:Triplet GUC at 1742 of hTERT mRNA was chosenas the cleavage site of the ribozyme.The designed ribozymewas comprised of 22nt catalytic core and 17nt flankingsequence.Computer-aided prediction suggested that theribozyme and hTERT mRNA could cofold into a properconformation.Endonuclease restriction and DNA sequencingconfirmed the correct insertion of the ribozyme gene intothe vector pGEMEX-1.CONCLUSION:This fundamental work of successfuldesigning and cloning of an anti-hTERT hammerheadribozyme has paved the way for further study of inhibitingtumor cell growth by cleaving hTERT mRNA with ribozyme.
AIM: To design a hammerhead ribozyme targeting human telomerase reverse transcriptase (hTERT) and clone it’s gene for future use in the study of tumor gene therapy. METHODS: Using the software RNA structure, the secondarystructure of hTERT mRNA was predicted and the cleavagesite of ribozyme was selected. A hammerhead ribozyme targetting this site was designed and bimolecular fold betweenthe ribozyme and hTERT was predicted. The DNA encoding the ribozyme was synthesized and cloned into pGEMEX-1 and the sequence of the ribozyme gene was confirmed by DNA sequencing .RESULTS: Triplet GUC at 1742 of hTERT mRNA was chosenas the cleavage site of the ribozyme.The designed ribozymewas comprised of 22nt catalytic core and 17nt flanking sequence. Computer-aided prediction suggested that the ribozyme and hTERT mRNA could cofold into a proper information. Endonuclease restriction and DNA sequencingconfirmed the correct insertion of the ribozyme gene intothe vector pGEMEX-1.CONCLUSION: This fundamental work of successfuld esigning and cloning of an anti-hTERT hammerhead ribozyme has paved the way for further study of inhibiting tumor cell growth by cleaving hTERT mRNA with ribozyme.