人心房肌AT1R和AT2R表达质粒的构建和鉴定  

Construction and identification of AT1R and AT2R expression plasmids from human atrial tissue

作　　者：余奕言 李涛 谭晓秋[1] 李妙龄[1] 

机构地区：[1]西南医科大学心血管医学研究所、医学电生理学省部教育部重点实验室、四川省心血管疾病防治协同创新中心,四川泸州646000

出　　处：《西南医科大学学报》2017年第1期31-34,共4页Journal of Southwest Medical University

基　　金：国家自然科学基金资助项目(81470022);泸州市-泸州医学院联合资助项目(2013LZLY-J23)

摘　　要：目的:血管紧张素Ⅱ(AngⅡ)作为肾素-血管紧张素系统(RAS)中重要的致心律失常的肽类物质,主要通过与细胞膜上AT1、AT2两种受体亚型结合而发挥其生物学作用。本研究构建带有荧光蛋白标记的AT1R和AT2R质粒,旨在为进一步观察其生物学效应奠定基础。方法:通过PCR扩增出含有Bam HⅠ和Hind Ⅲ酶切位点的目的基因(AT1R、AT2R)片段,然后分别与有着相同酶切位点的载体pDs Red-N1、pEGFP-N3连接。将重组子转入TOP10感受态细胞中,通过抗生素筛选、酶切鉴定等方法挑选出阳性克隆,并经测序鉴定。结果:经过定向克隆的方法,最后得到的表达质粒酶切结果正确,测序结果与基因库数据基本一致。结论:成功构建带有红色荧光表达人心房肌AT1R、AT2R的质粒pDs Red-N1-AT1R、pDs Red-N1-AT2R和带有绿色荧光表达人心房肌AT1R、AT2R的质粒pEGFP-N3-AT1R、pEGFP-N3-AT2R。Objective： Angiotensin II （Ang II）, an important molecule in the renin-angiotensin system （RAS）, mainly binds to AT1 and AT2 receptors to achieve its biological effect. This research aimed to construct AT1 and AT2 expression plasmids to further study their biological functions. Methods： Amplified by PCR with primers containing BamH I and Hind III restriction sites and with eDNA from human atrial tissue, and the PCR products of AT1R and AT2R were purified and cloned into pDsRed-N1 and pEGFP-N3 respectively, generating four plasmids. The recombinant plasmids were transformed into TOP10 competent cells, and positive clones were selected by kanamycin resistance and restriction digestion. Results： The identities of the four plasmids were confirmed by DNA sequencing. Conclusion： The expression plasmids, pDsRed-N1-AT1R, pDsRed-N1-AT2R, pEGFP-N3-AT1R, and pEGFP-N3-AT2R were constructed successfully.

关 键 词：血管紧张素II受体 质粒构建 肾素-血管紧张素系统 

分 类 号：R34[医药卫生—基础医学]