本文選題：Ⅰ型大麻素受體　切入點：逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)　出處：《中國藥房》2015年16期 　論文類型：期刊論文

【摘要】：目的:構(gòu)建大鼠Ⅰ型大麻素受體(CB1)真核基因表達(dá)載體,并檢測其在細(xì)胞中的表達(dá)情況。方法:從大鼠海馬組織中提取總RNA,逆轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)擴增出CB1基因片段,產(chǎn)物連接到p MD18-T載體上。陽性實驗篩選出陽性克隆,經(jīng)Eco RⅠ、Bam HⅠ雙酶切及測序鑒定后,將其連接到載體pc DNA 3.1(+)中,構(gòu)建質(zhì)粒載體pc DNA3.1(+)-CB1,脂質(zhì)體轉(zhuǎn)染原代人胚腎293細(xì)胞(HEK293細(xì)胞)。用Western blot實驗鑒定細(xì)胞中CB1蛋白的表達(dá),用免疫熒光細(xì)胞染色聯(lián)合激光掃描共聚焦法檢測其在細(xì)胞中表達(dá)的部位。結(jié)果:采用RT-PCR成功獲得大鼠CB1基因,PCR擴增得到1 500 bp左右的CB1基因片段,雙酶切鑒定及DNA測序證實質(zhì)粒載體pc DNA3.1(+)-CB1構(gòu)建成功。Western blot實驗、免疫熒光細(xì)胞染色聯(lián)合激光掃描共聚焦法證實載體pc DNA3.1(+)-CB1能在HEK293細(xì)胞中表達(dá),且表達(dá)產(chǎn)物主要分布在細(xì)胞膜表面和細(xì)胞質(zhì)中。結(jié)論:構(gòu)建的pc DNA3.1(+)-CB1表達(dá)載體能成功轉(zhuǎn)入真核HEK293細(xì)胞,并在細(xì)胞膜表面和細(xì)胞質(zhì)中表達(dá)CB1蛋白。
[Abstract]:Objective: to construct the eukaryotic expression vector of rat type I cannabinoid receptor (CB1) and to detect its expression in the cells. Methods: total RNAs were extracted from rat hippocampal tissue and CB1 gene fragments were amplified by reverse transcription-polymerase chain reaction (RT-PCR). The product was ligated to p MD18-T vector. The positive clone was screened out by positive experiment. After double digestion and sequencing of Eco R 鈪，

本文編號：1576185