抗Caspase-3核酶的体外制备与活性鉴定  

Preparation and identification of hammerhead ribozyme in vitro against rat caspase-3 mRNA fragment

作　　者：徐人欢[1] 周霞秋[1] 谢青[1] 金由辛 廖丹[1] 

机构地区：[1]上海第二医科大学附属瑞金医院传染科,上海200025

出　　处：《中华肝脏病杂志》2000年第6期361-363,共3页Chinese Journal of Hepatology

基　　金：上海市高等学校青年科学基金!(98QN60);中国科学院重大项目基金!(KJ951-B1-610)

摘　　要：目的：研究针对大鼠凋亡基因Caspase-3设计的核酶（Rz107和Rz544）的体外转录，切割及活性鉴定。方法：大鼠Caspase-3基因的PCR片段克隆于T载体T7 启动子下游，32P标记的体外转录物作为靶RNA。设计合成并克隆针对大鼠Caspase-3 mRNA的核酶（Rz107和Rz544），32P标记转录,核酶与靶RNA按一定比例进行体外切割实验。 结果：Rz107 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℃。其米氏常数（Km）为14.13nmol/L, 酶催化反应速度常数（kcat）为2.31/min。而Rz544则无切割活力。 结论：体外制备的Rz107具有良好的特异催化切割活性，它有望通过切割Caspase-3而抑制凋亡发生，可发展为新一代抗肝内炎症的核酸药物。Objective: To study the transcript effect and cleavage activity in vitro of rat Caspase-3 specific ribozyme (Rz107 and Rz544). Methods: Rat caspase-3 gene fragment was cloned into T-vector under the control of T7 promoter. 32P-labeled caspase-3 transcript was target-RNA. Rz107 and Rz544 genes against caspase-3 mRNA were cloned and transcribed in vitro. Cleavage reaction was detected. Results: It was found that Rz107 was active at 37℃ and more so at higher temperature within allowing temperature range. The optimal temperature was 50℃. For Rz107, Km and kcat was 14.13 nmol/L and 2.31 min-1, respectively. However, the Rz544 had no cleavage activity at all. Conclusion: Rz107 prepared in vitro possesses the perfect specific catalytic cleavage activity. It is hopeful that Rz107 would be developed to be a new nucleic acid drug that could effectively inhibit the inflammation of hepatitis through cleaving the key gene, caspase-3, in apoptosis in vivo.

关 键 词：RNA 活性鉴定 抗Caspase-3S核酸 肝细胞凋亡 

分 类 号：R392[医药卫生—免疫学]