摘要
目的构建cerbB2特异性核酶(ribozyme)基因及其底物基因的体外转录载体并探讨其体外切割作用。方法在计算机设计的核酶RZ1基因的3′端加上新的限制性酶切位点EcoRV,先合成RZ1基因并将之与其底物分别克隆入体外转录载体pGEM3Zf(-),经用EcoRV快速初选出含有RE1基因重组子并测序鉴定。体外转录物用32P标记。体外切割作用在Mg++的存在下,37℃反应1小时,进行聚丙烯酰胺凝胶电泳,放射自显影后经图像分析计算出切割率。结果用EcoRV筛选很快鉴定出了含有RE基因的重组子并命名为pGEM3ZRZ1,自动测序证实合成的核酶基因序列正确并已被准确克隆入pGEM3Zf(-)的T7启动子。RZ1的靶基因片段也被插入pGEM3Zf(-)的SP6启动子下。经体外转录出核酶RZ1及靶RNA后,体外切割反应1小时,79.3%的靶RNA即被切开。结论cerbB2特异性核酶RZ1在体外有良好的切割活性,该实验为进一步研究其在肿瘤基因治疗中的作用奠定了基础。
Objective To construct in vitro transcription vectors of genes of c erbB 2 specific ribozyme and its substrate and to probe its in vitro cleavage action. Methods According to the computer design, a specific restriction site EcoR V was added to the 3′ end of the ribozyme gene (RZ1). Then, the RZ1 gene and its substrate gene were cloned into the in vitro transcription vector pGEM3Zf(-) separately. The recombinants containing RZ1 gene were first screened by agrose ge1 electrophoresis through EcoR V digestion and was identified by automatic sequencing. The products of in vitro transcription were labeled with 32 P. In vitro cleavage reaction was performed at 37℃ for 1h under the presence of Mg ++ . The cleavage product was analyzed by polyacrilamide gel electrophoresis. After autoradiography, the cleavage rate was counted by image analysis. Results The recombinants containing the RZ1 gene were successfully selected by the EcoR V digestion and were designated as pGM3Z RZ1. The automatic sequence analysis proved that the synthesized RZ1 gene was correct. The target gene was also cloned into the pGEM3Zf(-) under SP6 promoter. After in vitro transcription, the cleavage reaction was shown to have cut off 79.3% target RNA in 1h. Conclusion The c erbB 2 oncogene specific ribozyme has a high activity in vitro. It lays a foundation for the study of the therapeutic use of ribozyme in gene therapy of cancer.
出处
《中华肿瘤杂志》
CAS
CSCD
北大核心
1999年第1期7-9,共3页
Chinese Journal of Oncology
基金
国家自然科学基金