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目的 体外研究针对大鼠凋亡基因Caspase 3设计的核酶 (Rz10 7)的体外转录 ,切割及活性鉴定及其在大鼠肝细胞系BRL 3A细胞中的切割活性。方法 将大鼠Caspase 3基因的PCR片段克隆于T载体T7启动子下游 ,32 P标记的体外转录物作为靶RNA。设计并合成针对于大鼠Caspase 3的核酶 (Rz10 7) ,将Rz10 7基因克隆于自我切割核酶载体p1 5的 5’ cis 核酶和 3’ cis 核酶之间 ,同样进行32 P标记转录。核酶与靶RNA按一定比例和条件保温切割 ,电泳后放射自显影。将携带有Rz10 7基因的真核表达质粒pcDNA3 1 Rz10 7电穿孔至BRL 3A细胞中 ,通过RT PCR方法分析Rz10 7的细胞内剪切活性。结果 Rz10 7在 37℃有活性。在一定温度范围内 ,其切割效率随温度升高而升高 ,最适温度为 50℃。其Km 为14 13nmol/L ,kcat为 2 31min 1。在BRL 3A细胞中 ,Rz10 7同样具有切割活性 ,其切割效率为 37%。结论 针对caspase 3mRNA设计的核酶 -Rz10 7,体外制备具有良好的特异催化切割活性 ,在体外及BRL 3A细胞内均能有效剪切底物RNA。这不仅对研究凋亡的发生及其机制提供了有利的研究工具 ,而且可通过抑制凋亡而成为治疗一些肝脏疾病的新方法。
OBJECTIVE: To study in vitro transcription, cleavage and activity of a ribozyme (Rz107) designed against rat apoptotic gene Caspase 3 and its cleavage activity in rat hepatocyte cell line BRL 3A. Methods The PCR fragment of rat Caspase 3 gene was cloned downstream of the T vector T7 promoter and the 32 P labeled in vitro transcript was used as target RNA. The ribozyme (Rz107) targeting rat Caspase 3 was designed and synthesized, and the Rz107 gene was cloned between 5’-cis ribozyme and 3’-ribozyme of self-cleaving ribozyme vector p15 Transcription. Ribozyme and target RNA by a certain percentage and conditions of thermal cutting, autoradiography after electrophoresis. The eukaryotic expression plasmid pcDNA3 1 Rz10 7 carrying the Rz10 7 gene was electroporated into BRL 3A cells and the intracellular shear activity of Rz10 7 was analyzed by RT PCR method. Results Rz10 7 is active at 37 ° C. In a certain temperature range, the cutting efficiency increases with increasing temperature, the optimum temperature is 50 ℃. The Km was 14 13 nmol / L and kcat was 2 31 min 1. Rz107 also has cleavage activity in BRL 3A cells with a 37% cut efficiency. Conclusion The ribozyme-Rz107 designed for caspase 3 mRNA has good specific catalytic cleavage activity in vitro and can effectively cleave substrate RNA in vitro and in BRL 3A cells. This not only provides a favorable research tool for studying the occurrence and mechanism of apoptosis, but also becomes a new method to treat some liver diseases by inhibiting apoptosis.