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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 eukaryotic expression plasmid of human cannabinoid receptor 1 (hCBⅠ) gene GV230 and express it in HEK-293 cells. Methods The total RNA of human cortical cells was used as a template to amplify the hCBⅠ gene. The recombinant plasmid was cloned into the eukaryotic expression vector GV230. The recombinant plasmid was named as GV230-hCBⅠ. The positive clones were screened and transiently transfected into HEK293 cells. The cells were observed under inverted fluorescence microscope. The expression of hCBⅠ gene in HEK293 cells was detected by confocal laser scanning microscopy (CLSM) and Western blot. Results The recombinant plasmid GV230-hCBⅠ was identified by double enzyme digestion and sequencing and was correctly constructed. After 48 h of transfection, the transfection rate was about 40%. The CB Ⅰ protein was mainly distributed in the cell membrane and expressed at specific molecular weight of about 50,000. Conclusion The recombinant eukaryotic expression plasmid GV230-hCBⅠ was successfully constructed and expressed in HEK293 cells, which laid the foundation for further study on the biological function of CB Ⅰ.