Study on the cleavage activity of U1 small nuclear RNA chimeric ribozyme against HCV RNA in vitro / 中华肝脏病杂志
Chinese Journal of Hepatology
;
(12): 749-751, 2004.
Article
in Chinese
| WPRIM
| ID: wpr-233673
ABSTRACT
<p><b>OBJECTIVE</b>To study the cleavage activity on the HCV RNA of a chimeric recombinant of HCV specific ribozyme and U1 small nuclear RNA, which compartmentalizes within the nucleolus.</p><p><b>METHODS</b>The third stem-loop sequence of human U1 snRNA (position 95-116) within pBSIISK+ U1 was substituted by hammerhead ribozyme against HCV RNA by PCR and cloning methods, and the constructed plasmid was named pBSIISK+ (U1-Rz). Then the whole gene fragment of the chimeric ribozyme was cloned into a pGEM-T vector under the control of T7 promoter, and the constructed plasmid was named pGEM- (U1-Rz). The pGEM- (U1-Rz) and pGEM-Rz (containing the same ribozyme sequence as that in U1-Rz) transcripts as enzyme were transcribed in vitro. Also the (32)P-labeled pCMV/T7-NCRC luc (containing the gene sequence of the whole 5'-NCR and part core of HCV RNA) transcripts as target-RNAs were transcribed in vitro. The enzymes were incubated with the target RNAs under different conditions and autoradiographed after denaturing gel-electrophoresis.</p><p><b>RESULTS</b>The sequencing result showed that the construction of U1 snRNA chimeric ribozyme was correct. Compared with the ribozyme alone, both of them were active at 37 degree C and with Mg2+ (10 mmol/L) and TrisCl (10 mmol/L, pH7.9), and there was no remarkable difference between them. The cleavage activity of the chimeric ribozyme increased with the prolongation of reaction time and increment of enzyme concentration.</p><p><b>CONCLUSION</b>Both ribozyme and U1 snRNA chimeric ribozyme exhibited specifically catalytic activity against HCV RNA in vitro. There was no remarkable difference between their cleavage efficiencies.</p>
Full text:
Available
Index:
WPRIM (Western Pacific)
Main subject:
Pharmacology
/
Therapeutics
/
Recombinant Fusion Proteins
/
RNA, Viral
/
Genetic Therapy
/
RNA, Small Nuclear
/
Chimera
/
RNA, Catalytic
/
Hepatitis C
/
Hepacivirus
Language:
Chinese
Journal:
Chinese Journal of Hepatology
Year:
2004
Type:
Article
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