大鼠CB1基因真核表达载体的构建及初步鉴定

发布时间：2018-03-06 19:34

  本文选题：Ⅰ型大麻素受体　切入点：逆转录-聚合酶链反应　出处：《中国药房》2015年16期 　论文类型：期刊论文

【摘要】：目的:构建大鼠Ⅰ型大麻素受体(CB1)真核基因表达载体,并检测其在细胞中的表达情况。方法:从大鼠海马组织中提取总RNA,逆转录-聚合酶链反应(RT-PCR)扩增出CB1基因片段,产物连接到p MD18-T载体上。阳性实验筛选出阳性克隆,经Eco RⅠ、Bam HⅠ双酶切及测序鉴定后,将其连接到载体pc DNA 3.1(+)中,构建质粒载体pc DNA3.1(+)-CB1,脂质体转染原代人胚肾293细胞(HEK293细胞)。用Western blot实验鉴定细胞中CB1蛋白的表达,用免疫荧光细胞染色联合激光扫描共聚焦法检测其在细胞中表达的部位。结果:采用RT-PCR成功获得大鼠CB1基因,PCR扩增得到1 500 bp左右的CB1基因片段,双酶切鉴定及DNA测序证实质粒载体pc DNA3.1(+)-CB1构建成功。Western blot实验、免疫荧光细胞染色联合激光扫描共聚焦法证实载体pc DNA3.1(+)-CB1能在HEK293细胞中表达,且表达产物主要分布在细胞膜表面和细胞质中。结论:构建的pc DNA3.1(+)-CB1表达载体能成功转入真核HEK293细胞,并在细胞膜表面和细胞质中表达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，

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