人源大麻素Ⅰ型受体基因真核表达质粒的构建及表达  

Construction of eukaryotic expression vector for human cannabinoid receptor 1 gene and expression of target protein

作　　者：郝坡[1] 马强[1] 孙厚良[1] 赖国旗[2] 李晶[3] 

机构地区：[1]重庆三峡医药高等专科学校,重庆404120 [2]重庆医科大学,重庆400016 [3]重庆三峡中心医院,重庆404000

出　　处：《中国生物制品学杂志》2016年第9期918-921,共4页Chinese Journal of Biologicals

基　　金：重庆市基础与前沿研究计划项目(cstc2014jcyjA10049);重庆市高等学校青年骨干教师资助计划

摘　　要：目的构建人源大麻素Ⅰ型受体(human cannabinoid receptor 1,hCBⅠ)基因GV230真核表达质粒,并于HEK-293细胞中进行表达。方法以人脑皮质细胞的总RNA为模板,扩增获得hCBⅠ基因,克隆至真核表达载体GV230,构建重组表达质粒GV230-hCBⅠ,筛选阳性克隆,脂质体瞬时转染HEK293细胞。置倒置荧光显微镜下观察,并采用激光共聚焦显微镜(confocal laser scanning microscope,CLSM)和Western blot法检测hCBⅠ基因在HEK293细胞中的表达。结果重组表达质粒GV230-hCBⅠ经双酶切及测序鉴定,构建正确。转染48 h后,转染率约40%,CBⅠ蛋白主要于细胞膜分布及表达,且于相对分子质量约50 000处可见特异性条带。结论成功构建了重组真核表达质粒GV230-hCBⅠ,并于HEK293细胞中表达,为进一步研究CBⅠ的生物学功能奠定了基础。Objective To construct the GV230 eukaryotic expression vector for human cannabinoid receptor 1（hCBⅠ）gene and express in HEK293 cells. Methods Full-length hCB1 cDNA was amplified by RT-PCR using the total RNA isolated from human brain gliacyte cells as a template, and cloned into eukaryotic expression vector GV230. HEK293 cells were transfected transiently with the positive clones of constructed recombinant plasmid GV230-hCB Ⅰ, and observed under inverted fluorescent microscope. The expression of hCB Ⅰ in HEK293 cells was identified by confocal laser scanning microscope（CLSM） and Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid GV230-hCB Ⅰ was constructed correctly. The transfection efficacy of HEK293 cells 48 h after transfection was about 40%. CB Ⅰ protein was mainly distributed in cell membrane, of which the relative molecular mass was about 50 000. Conclusion Recombinant eukaryotic expression vector GV230-hCBⅠ was successfully constructed and expressed in HEK293 cells, which laid a foundation of further study on the biological function of CBⅠ.

关 键 词：人源大麻素Ⅰ型受体 真核细胞 基因表达 

分 类 号：Q782[生物学—分子生物学]