Cleavage activity of anti-transforming growth factor ?_1 RNA by U1 snRNA chimeric ribozyme in vitro / 中国病理生理杂志

ABSTRACT

AIM: To determine the cleavage activity of anti-transforming growth factor ?1 hammerhead ribozymes which was inserted into U1 small nuclear RNA in cell-free system.METHODS: The hammerhead ribozyme targeting against transforming growth factor ?1 was designed through the analysis of computer software.The ribozyme fragments were synthesized and cloned into the U1 snRNA ribozyme vector pZeoU1EcoSpe,which contained U1 snRNA promoter/enhancer and terminator.TGF ?1 cDNA partial fragment was generated by RT-PCR,and then cloned into the T-vector at the downstream of T7 promoter.The transcripts of ribozyme and target RNA incorporated into isotope were transcribed in vitro and purified by denaturing polyacrylamide gel electrophoresis.-labeled U1 snRNA chimeric ribozyme transcripts were incubated with target-RNAs at different conditions and autoradiographed after running denaturing PAGE.RESULTS: U1snRNA chimeric ribozyme(U1Rz803) cleaved TGF?1 mRNA efficiently and specifically at 37 ℃,while the disable ribozyme(U1Rz803m) showed no cleavage activity,so these indicated the design of U1Rz803 was correct.CONCLUSION: U1Rz803 prepared in this study possesses the perfect specific catalytic cleavage activity in cell-free system.These results indicate that U1 snRNA chimeric ribozyme U1Rz803 may suppress the expression of TGF?1 in vivo,therefore it may provide a new means for exploring the role of TGF?1 in hematopoietic regulation in the future.