摘要
目的:在中国仓鼠卵巢(CHO)细胞上稳定转染人重组阿片受体样受体(hORL1),为体外高通量筛选hORL1受体作用药物及相关药物分子机制研究奠定基础。方法:用脂质体介导法将pcDNA3.1(+)-hORL1重组质粒稳定转染到缺乏hORL1的CHO细胞中,然后用含G418的选择性培养液进行筛选,挑取耐药克隆;用放射性配体-受体结合实验进一步筛选阳性克隆,对阳性克隆受体亲和力和表达量进行分析,用[35S]GTPγS结合实验分析表达受体的功能。结果与结论:在稳定转染hORL1的克隆细胞中,[3H]伤害感受肽的Kd和Bmax值分别为(0.44±0.21)nmol/L和(0.35±0.06)pmol/mg蛋白。伤害感受肽刺激受体结合[35S]GTPγS的EC50值为3.45nmol/L。以上结果与文献报道的天然hORL1受体的特性相似,说明该细胞株表达的hORL1受体与天然的hORL1受体具有基本一致的生物学特性,证实成功建立了稳定表达人阿片受体样受体的细胞模型。
Objective：To stably transfect human opioid receptor-like 1 （ hORL1 ） in Chinese hamster ovary（CHO） cells. Methods：pcDNA3.1 （ ＋ ）-hORL1 was stably transfected into CHO cells by a lipofectamine based method. Transfected CHO cells were selected in culture medium containing G418. Radioligand receptor binding assay and [ 35S] GTPγS binding assay were used to determine densities and functions of the expressed receptors. Results and Conclusion： hORL1 was expressed in the CHO cells. The Kd and Bmax were （0.44±0.21 ） nmol/L and （0.35±0.06） pmol/mg protein, respectively, for hORL1 in [ 3H] nociceptin binding assay. The EC50 of nociceptin was 3.45 nmol/Lin stimulation of [^35S] GTPγS binding by nociceptin. A model system of CHO cells with stable expression of hORL1 is established.
出处
《军事医学科学院院刊》
CSCD
北大核心
2009年第5期409-411,485,共4页
Bulletin of the Academy of Military Medical Sciences
基金
军队"十一五"青年学者项目(06Q075)
