摘要
目的:研究抗转化生长因子β1U1snRNA嵌合型锤头状核酶的细胞外切割活性。方法:通过计算机设计针对TGFβ1的锤头状核酶,然后把合成的核酶片段克隆入含有U1 snRNA启动子/增强子和终止子的U1snRNA核酶载体中。通过RT-PCR扩增获得TGFβ1的部分基因片段,将其克隆入T载体中T7启动子的下游,体外转录获得核酶和靶RNA,转录过程中掺入同位素,通过变性的聚丙烯酰胺凝胶电泳纯化回收。[32P]标记的核酶与靶RNA在不同条件下进行切割反应,变性PAGE电泳,放射自显影,分析反应结果。结果:活性的U1 snRNA嵌合型核酶(U1Rz803)在生理温度下具有良好特异的切割活性;而点突变型核酶U1Rz803m没有切割活性,因此这些结果显示U1Rz803设计是正确的。结论:本研究中制备的U1Rz803具有良好的特异催化切割活性。U1 snRNA嵌合型核酶U1Rz803有望在胞内抑制TGFβ1的表达,为研究转化生长因子(TGF)β1在造血调控中的作用机制提供有效工具。
AIM： To determine the cleavage activity of anti - transforming growth factor β1 hammerhead ribozymes which was inserted into U1 small nuclear RNA in cell -free system. METHODS： The hammerhead ribozyme targeting against transforming growth factor β1 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 β1 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. [ ^32 p] - labeled U1 snRNA chimeric ribozyme transcripts were incubated with target - RNAs at different conditions and autoradiographed after running denaturing PAGE. RESULTS： UlsnRNA chimeric ribozyme （U1Rz803 ） cleaved TGFβ1 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 TGFβ1 in vivo, therefore it may provide a new means for exploring the role of TGFβ1 in hematopoietic regulation in the future.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2007年第8期1609-1612,共4页
Chinese Journal of Pathophysiology
关键词
转化生长因子Β
RNA
催化
U1小核RNA嵌合酶
Transforming growth factor beta
RNA, catalytic
U1 small nuclear RNA chimeric ribozyme
