论文部分内容阅读
目的 设计针对小鼠内质网应激凋亡途径中Caspase-12 mRNA的锤头状核酶(Rz138、Rz218),通过靶基因及核酶的体外转录、切割,进行核酶活性鉴定,评估其应用前景。 方法 小鼠Caspase-12基因的逆转录聚合酶链反应(RT-PCR)扩增片段克隆于PGEM-T载体的T7启动子下游,通过α-32P UTP标记的体外转录物作为靶RNA。设计合成针对小鼠Caspase-12 mRNA的核酶,通过PCR方法扩增核酶的转录模板,采用非同位素标记法行体外转录,核酶与靶RNA进行体外切割实验。 结果 在37℃,Rz138、Rz218均有切割活性,Rz138的切割效率较高,几乎100％。 结论 Rz138在体外具有良好的特异催化切割活性,它有望通过切割Caspase-12 mRNA而抑制内质网应激介导的细胞凋亡发生。
Objective To design a hammerhead ribozyme (Rz138, Rz218) targeting Caspase-12 mRNA in the apoptotic pathway of endoplasmic reticulum stress in mice and identify its ribozyme activity by in vitro transcription and cleavage of target genes and ribozymes Application prospects. Methods Mouse Caspase-12 gene was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR). The amplified fragment was cloned downstream of the T7 promoter of PGEM-T vector and labeled with α-32P UTP labeled in vitro transcripts as target RNA. The ribozyme targeting mouse Caspase-12 mRNA was designed and synthesized. The transcriptional template of ribozyme was amplified by PCR. The in vitro transcription of ribozyme and target RNA was performed by using non-isotope labeling method. Results Both Rz138 and Rz218 had cleavage activity at 37 ° C, and the cleavage efficiency of Rzl38 was almost 100%. Conclusion Rz138 has good specific catalytic activity in vitro. It is expected that Rz138 could inhibit endoplasmic reticulum stress-induced apoptosis by cleaving Caspase-12 mRNA.