mTOR反义RNA真核表达载体的构建及其在血管平滑肌细胞中的表达  

Construction of eukaryotic expression vector of mTOR gene antisense RNA and its expression in vascular smooth muscle cells

作　　者：胡新华[1] 杨军[1] 张志深[1] 刘程伟[1] 刘戈飞[1] 张强[1] 

机构地区：[1]中国医科大学附属第一医院血管外科,辽宁沈阳110001

出　　处：《中国医科大学学报》2006年第4期337-339,共3页Journal of China Medical University

基　　金：国家自然科学基金资助项目(30400435;30371401);辽宁省博士启动基金资助项目(20041053)

摘　　要：目的:构建哺乳类动物雷帕霉素靶蛋白(mTOR)的反义RNA真核表达载体,观察其对血管平滑肌细胞(VSMC)功能的影响。方法:提取人VSMC总RNA,反转录-聚合酶链反应(RT-PCR)扩增mTOR基因cDNA序列,经pGEM-T载体克隆后双酶切,将cDNA序列反向插入绿色荧光蛋白表达载体pEGP-C1,构建mTOR基因反义RNA真核表达载体。转染VSMC,采用W estern b lot法检验反义表达载体对mTOR蛋白表达的影响,流式细胞仪检测细胞周期的变化。结果:经RT-PCR获得664 bp产物,T载体克隆后,经DNA测序,确定该片段为mTOR基因cDNA,进而构建反义RNA真核表达载体pEGFP-C1-mTOR,测序证明序列正确后转染VSMC,证实其能够显著抑制mTOR蛋白产物表达,VSMC的分裂、增殖过程受阻。结论:已经成功构建mTOR基因的反义RNA真核表达载体。Objective： To construct pEGFP-C1-mTOR vector, an eukaryotic expression plasmid of mTOR gene antisense RNA, and to study the effect of this vector on the function of vascular smooth muscle cells （VSMC）. Methods： The conservative region of mTOR gene was amplified by revcrse transcriptase polymerase chain reaction （RT-PCR） after total RNA was extracted from human VSMC and then cloned into pGEM-T vector. After the recombinant plasmid was certified by DNA sequencing, the conservative region of mTOR gene was inserted into pEGFP-C1 vector reversedly, and pEGFP-C1- mTOR vector was constructed. The efficiency of antisense inhibition was verified by Western blotting after the transfection of VSMC. The cell cycle was determined by flow cytometry. Results： A 664- bp fragment including conservative region of roTOR gene was obtained by RT-PCR. After cloned by pGEM-T vector and certified by DNA sequencing, pEGP-C1-mTOR vector was successfully constructed by recombinant DNA technology. Additionally, pEGFP- C1- mTOR vector could efficiently decrease mTOR protein expression level by 82% 72 hours after the transfection of VSMC. The cell cycle was stunned. Conclusion： The efficient expression vector of roTOR gene antisense RNA, pEGFP-C1- mTOR , has been constructed successfully.

关 键 词：雷帕霉素靶蛋白 反义技术 基因表达载体 

分 类 号：Q753[生物学—分子生物学]