编码大鼠PRMT1基因shRNA质粒的构建和鉴定  

Construction and Identification of Plasmid pGenesil-1-shRNA

作　　者：方雅琴[1] 邱龄[2] 肖传实[2] 焦延景[1] 张圣雪[1] 

机构地区：[1]山西医科大学 [2]山西医科大学第二医院

出　　处：《中西医结合心脑血管病杂志》2008年第3期299-302,共4页Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease

摘　　要：目的研究蛋白精氨酸甲基转移酶1(PRMT1)基因表达的改变与血管内皮功能及动脉粥样硬化的关系。方法构建PRMT1短发夹RNA(shRNA)质粒。在GenBank中查到大鼠PRMT1基因mRNA序列并输入siRNA网上设计软件OptiRNA中,选取两条靶序列,设计并合成编码shRNA序列的DNA单链,经退火连接后,与双酶切线性化处理回收的pGenesil-1质粒载体连接,用含卡那霉素抗性的LB平板筛选阳性克隆,小提质粒后行酶切鉴定并测序。结果构建的大鼠PRMT1-shRNA质粒经酶切鉴定及测序与预期相符。结论本研究结果为进一步研究基因干扰PRMT1基因对血浆同型半胱氨酸(Hcy)、非对称性二甲基精氨酸(ADMA)水平及血管内皮功能的影响奠定了基础。Objective To construct the expression plasmids of short hairpin RNA （shRNA） targeted to the rat protein arginine methyltransferases 1 （PRMT1） gene for investigating the relationship between the expression of PRMT1 gene and the function of vascular endothelium, Methods The sequence of rat PRMT1 mRNA was found in the GenBank and copied to the design software at net. Two target sequences were selected, Single strands of encoding shRNA were designed, synthesized and conjuncted by annealing, after which the annealed duplexes were conjugated with plasmid vectors pGenesil - 1 that were on linearization through digested with BamH I and Hind m enzyme, generating the constructed pGenesil - 1 - PRMT1 - shRNA, After these plasmids were extracted, they were identified with the digestion of Sal Ⅰ enzyme determined sequence, and the inserted sequences were carried on DNA sequencing. Results The constructs were all verified to fulfil design requirements through digested with Sal Ⅰ enzyme. The inserted sequences were verified by DNA sequencing, The recombined shRNA plasmids was certified to be in the right rank, Conclusion This experiment laid the foundation for further research on the effect of RNA interfere of PRMT1 gene on the plasma levels of homocysteine and asymmetric methytarginine and the function of vascular endothelium.

关 键 词：蛋白精氨酸甲基转移酶1 短发夹RNA 基因干扰 同型半胱氨酸 非对称性二甲基精氨酸 动脉粥样硬化 

分 类 号：R541.4[医药卫生—心血管疾病]