抗半胱氨酸蛋白酶-7核酶的克隆、体外表达与切割活性的研究  

Clone, expression and cleavage activity of anti-caspase-7 hammerhead ribozyme in vitro

作　　者：张(韦华)[1] 谢青[1] 周霞秋[1] 姜山[1] 金由辛[2] 

机构地区：[1]第二医科大学附属瑞金医院感染科,上海200025 [2]中国科学院上海生物化学研究所

出　　处：《中华肝脏病杂志》2004年第11期684-687,共4页Chinese Journal of Hepatology

基　　金：国家自然科学基金(30170850)

摘　　要：目的 设计并合成抗小鼠半胱氨酸蛋白酶-7(caspase-7)的锤头状核酶,并对它们的体外表达与体外切割活性进行研究。 方法 采用计算机软件对锤头状核酶和小鼠caspase-7基因的二级结构进行分析,核酶的DNA序列在体外由自动合成仪合成,小鼠caspase-7目的基因通过RT-PCR扩增获得,将核酶基因和caspase-7基因分别克隆入载体pBSKneoU6’和pGEM-T中,通过体外转录得到核酶和caspase-7mRNA,通过体外切割筛选出具有切割活性的核酶。 结果 针对目的基因的333和394位点发计合成了两个核酶:Rz333和Rz394。成功获得caspase-7 mRNA的表达载体pC7及含有核酶的重组质粒pU6Rz333和pU6Rz394,通过体外转录表达出含有caspase-7 mRNA的987 nt的靶mRNA及完整的核酶Rz333(47 nt)和Rz394(50 nt)。体外切割实验证实Rz333能够定点切割caspase-7 mRNA,产生243 nt和744 nt的切割产物,切割效率为67.98％。Rz394不能切割靶基因。 结论 核酶Rz333能够位点特异性的切割caspase-7 mRNA。Objective To design hammerhead ribozymes against mouse caspase-7 and to study their expression and cleavage activity in vitro. Methods The secondary structures of ribozyme and caspase-7 genes were analyzed and simulated by computer. Ribozymes DNA sequences were synthesized by automatic synthetic apparatus. Caspase-7 DNA sequence was acquired by reverse transcription PCR. Ribozymes and caspase-7 DNA sequences were separately cloned into pBSKneo U6 and pGEM-T vectors. Ribozymes and caspase-7 mRNA were obtained by transcription in vitro, and ribozymes cleavage activity was identified by cleavage experiment in vitro. Results Two ribozymes named Rz333 and Rz394 targeting 333 and 394 sites in caspase-7 mRNA were designed by computer software, and their DNA sequences were synthesized. The expression vector of caspase-7 and plasmids containing Rz333 and Rz394 were reconstructed successfully. Ribozymes and caspase-7 mRNA were expressed by in vitro transcription. In vitro cleavage experiments showed that Rz333 cleaved caspase-7 mRNA and produced 243nt and 744nt segments. The cleavage efficiency is 67.98%, while Rz394 cannot cleave caspase-7 mRNA. Conclusions Rz333 can site-specifically cleave caspase-7 mRNA.

关 键 词：抗半胱氨酸蛋白酶-7 基因克隆 基因表达 切割活性 小鼠 锤头状核酶 

分 类 号：R341[医药卫生—基础医学]