Preparation and identification of anti-transforming growth factor beta1 U1 small nuclear RNA chimeric ribozyme in vitro.
World J Gastroenterol
; 9(3): 572-7, 2003 Mar.
Article
em En
| MEDLINE
| ID: mdl-12632521
ABSTRACT
AIM:
To study the preparation and cleavage activity of anti-transforming growth factor (TGF)beta1 U1 small nuclear (sn) RNA chimeric hammerhead ribozymes in vitro.METHODS:
TGFbeta1 partial gene fragment was cloned into T-vector at the downstream of T7 promoter. (32)p-labeled TGFbeta1 partial transcripts as target RNA were transcribed in vitro and purified by denaturing polyacrylamide gel electrophoresis (PAGE). Anti-TGFbeta1 ribozymes were designed by computer, then synthetic ribozyme fragments were cloned into the U1 ribozyme vector pZeoU1EcoSpe containing U1 snRNA promoter/enhancer and terminator. (32)p-labeled U1 snRNA chimeric ribozyme transcripts were gel-purified, incubated with target-RNAs at different conditions and autoradiographed after running denaturing PAGE.RESULTS:
Active U1snRNA chimeric ribozyme (U1Rz803) had the best cleavage activity at 50 degrees; at 37 degrees, it was active, K(m)=34.48 nmol/L, K(cat)=0.14 min(-1); while the point mutant ribozyme U1Rz803(m) had no cleavage activity, so these indicated the design of U1Rz803 was correct.CONCLUSION:
U1Rz803 prepared in this study possessed the perfect specific catalytic cleavage activity. These results indicate U1 snRNA chimeric ribozyme U1Rz803 may suppress the expression of TGFbeta1 in vivo, therefore it may provide a new avenue for the treatment of liver fibrosis in the future.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas Recombinantes de Fusão
/
RNA
/
RNA Nuclear Pequeno
/
RNA Catalítico
/
Fator de Crescimento Transformador beta
Tipo de estudo:
Diagnostic_studies
/
Prognostic_studies
Limite:
Animals
Idioma:
En
Revista:
World J Gastroenterol
Ano de publicação:
2003
Tipo de documento:
Article