【摘 要】
：
目的 构建人Ⅰ型大麻受体(CB1)基因GV230真核表达质粒,并检测hCB1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-hCB1,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(CLSM)和蛋白质印迹法(Western blot)
【机 构】
：
重庆三峡医药高等专科学校,重庆万州404120 重庆医科大学,重庆渝中400016
【出 处】
：
第十六届全国神经精神药理学学术会议
论文部分内容阅读
目的 构建人Ⅰ型大麻受体(CB1)基因GV230真核表达质粒,并检测hCB1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-hCB1,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(CLSM)和蛋白质印迹法(Western blot)检测hCB1基因表达产物在细胞的表达情况。结果 扩增出hCB1基因片段,通过酶切及测序鉴定证明成功构建了重组表达质粒,荧光显微镜观察和Western blot检测到目的蛋白在转染细胞中的表达,激光共聚焦显微镜观察到CB1受体在胞膜分布和表达。结论 成功构建GV230-hCB1质粒,该质粒在HEK293细胞中能表达CB1蛋白,此为进一步研究hCB1生物学功能奠定了实验基础。
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