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pRluc-hKOR-pcDNA3.1真核重组质粒的构建及在HEK293细胞中的表达 被引量：4

Construction of pRluc-hKOR-pcDNA3.1 recombinant eukaryotic expression vector and its expression in HEK293 cells
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摘要 目的:构建与海肾荧光素酶(renilla luciferase,Rluc)融合的人κ型阿片受体(kappa opioid receptor,KOR)的真核表达载体,用于生物发光共振能量转移法检测人KOR与其它受体间的相互作用。方法:以质粒pcD-NA3.1-hKOR为模板,PCR方法扩增人KOR。扩增的人KOR用NotⅠ和XhoⅠ双酶切,同时用这2种酶双酶切质粒pRluc-pcDNA3.1。然后将这2种酶切产物按常规方法连接、转化大肠杆菌Top10。挑取菌落培养,提取质粒,然后进行酶切鉴定,最后进行测序。将测序正确的重组载体用脂质体法转染人胚胎肾(human embryonic kidney293,HEK293)细胞,免疫荧光染色,共聚焦显微镜观察。结果:扩增出了1条约1.2kb的片段,与预期的人KOR大小相符,质粒酶切结果显示重组质粒pRluc-hKOR-pcDNA3.1被切成2条片段,其中1条为pRluc-pcDNA3.1载体大小,另1条为目的片段大小。经测序鉴定,序列与GenBank(NM_000912)中的序列高度同源。共聚焦显微镜观察显示,阿片受体主要表达在细胞膜上。结论:构建成功了pRluc-hKOR-pcDNA3.1重组真核表达载体,此表达载体可用于检测人KOR与其它受体之间的相互作用。
作者 李雅林 白波 陈京 刘有旺 刘海青 路海
机构地区 泰山医学院 山东农业大学 济宁医学院
出处 《中国病理生理杂志》 CAS CSCD 北大核心 2009年第10期2078-2080,共3页 Chinese Journal of Pathophysiology
基金 国家自然科学基金资助项目(No.30870932) 山东省自然科学基金资助项目(No.Y2005C47) 山东省科技公关计划资助项目(No.2006GG2202037) 山东省自然科学基金资助项目(No.Y2007D01)
关键词 受体 阿片样 κ 重组真核表达载体 HEK293细胞 Receptors, opioid, kappa Recombinant eukaryotic expression vector HEK293 cells
分类号 R331 [医药卫生—人体生理学]
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中国病理生理杂志
2009年 第10期

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