大鼠CB1基因真核表达载体的构建及初步鉴定  

Construction and Preliminary Identification of the Eukaryotic Expression Vector of CB1 Gene in Rats

作　　者：刘佳[1] 李晶[2] 严磊[3] 赵婷婷[3] 王慧娟[3] 赖国旗[3] 龙明[2] 

机构地区：[1]重庆三峡中心医院药学部,重庆404000 [2]重庆三峡医药高等专科学校第一附属医院神经外科,重庆404120 [3]重庆医科大学实验动物中心,重庆400016

出　　处：《中国药房》2015年第16期2184-2187,共4页China Pharmacy

基　　金：重庆市基础与前沿研究计划项目(No.cstc2014jcyj A10049);重庆市卫生局医学科研项目(No.2012-1-096);重庆市高等学校青年骨干教师资助计划

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。OBJECTIVE： To construct the eukaryotic expression vector of cannabinoid receptor 1 （CB1） in rats and test its ex- pression in cells. METHODS： The total RNA was extracted from the hippocampus tissues in rats, and subjected to reverse transcrip- tion polymerase chain reaction （RT-PCR） to produce CB1 gene segments by amplification. The product was connected to pMD18-T, and after identified by double enzyme digestion with EcoR I and BamH I and sequencing following the screening by the positive test, it was cloned in the carrier pcDNA 3.1（＋） to construct plasmid carrier pcDNA3.1（＋）-CB1 which was transfected into HEK293 cells （primary human embryo kidney 293 cells） by liposome. Western blot was adopted to determine the protein ex- pression of CB1 in the cells. Immunofluorescent cytochemical staining combined with confocal laser scanning was employed to de- termine the position of CB1 protein in the transfected cells. RESULTS： CB1 was successfully obtained in the RT-PCR. About 1 500 bp CB1 segments were produced by amplification in the PCR. The plasmid vector pcDNA3.1 （＋）-CB1, after the determination by double enzyme digestion and DNA sequencing, was confirmed to be successfully constructed. According to the identification by western blot and immunofluorescent cytochemical staining combined with confocal laser scanning, the carrier pcDNA 3.1 （＋）-CB1 can be expressed in HEK293 cells, and the expression products were mainly distributed in the cell membrane surface and matrix. CONCLUSIONS： CB1 protein can be expressed on the surface of the membrane and in the cytosol of HEK293 cells into which re- combinant plasmid pcDNA3.1 （＋）-CB l is transfected.

关 键 词：I型大麻素受体 逆转录.聚合酶链反应 质粒载体 

分 类 号：R965[医药卫生—药理学]