人Ⅱ型大麻受体真核表达体系构建及其在 HEK293细胞中的表达

摘要

目的：构建人Ⅱ型大麻受体（ hCB2）基因GV230真核表达质粒，并检测hCB2基因在HEK293细胞中的表达。方法利用人脑皮质细胞的总RNA为模板，RT-PCR获得cDNA，通过酶切、连接及测序鉴定正确后，再将目的片段插入真核表达载体GV230，构建重组表达质粒GV230-hCB2，阳性克隆用脂质体瞬时转染HEK293细胞。激光共聚焦扫描显微镜和Western blotting法检测hCB2基因表达产物在细胞的表达情况。结果扩增出hCB2基因片段，成功构建了重组表达质粒，并检测到目的蛋白在转染细胞中表达，观察到hCB2受体在胞膜分布和表达。结论成功构建GV230-hCB2质粒，该质粒在HEK293细胞中能表达hCB2蛋白，此为进一步研究hCB2生物学功能奠定了实验基础。%Objective To construct the eukaryotic expression plasmid of human cannabinoid receptor 2 ( hCB2) gene GV230 and to detect the expression of hCB2 gene in the HEK293 cells.Methods Full length of hCB2 cDNA was obtained by RT-PCR with total RNA isolated from human T lymphocytes, and then we inserted the target fragment into eukaryotic ex-pression vector GV230 to construct the recombinant expression plasmid GV230-hCB2 after enzyme digestion, connection and sequencing.We used the liposome to transiently transfect HEK293 cells.The expression of hCB2 gene expression products was detected by Western blotting and confocal laser scanning microscope (CLSM).Results The hCB2 gene fragments were amplified, and we successfully constructed the recombinant expression plasmid, detected the target protein expressing in the transfected cells, and observed the distribution and expression of hCB2 receptor in the cell membrane. Conclusion We successfully contrast the GV230-hCB2 plasmid expressing hCB2 protein in the HEK293 cells, which lays the experimental foundation for further research of hCB2 biology function.