论文部分内容阅读
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 recombinant opioid receptor-like receptor (hORL1) on Chinese hamster ovary (CHO) cells and lay the foundation for the high-throughput screening of hORL1 receptor drugs and related molecular mechanisms in vitro. Methods: The recombinant plasmid pcDNA3.1 (+) - hORL1 was stably transfected into CHO cells lacking hORL1 by liposome - mediated method and then screened with selective medium containing G418 to select resistant clones. Radioligand - receptor binding experiments were further screened for positive clones, positive clones receptor affinity and expression analysis, [35S] GTPγS binding assay analysis of receptor function. RESULTS AND CONCLUSION: The Kd and Bmax values of [3H] nociceptin were (0.44 ± 0.21) nmol / L and (0.35 ± 0.06) pmol / mg protein, respectively, in cloned cells stably transfected with hORL1. The nociceptive peptide stimulating receptor bound [35S] GTPγS had an EC50 value of 3.45 nmol / L. The above results are similar to those of the native hORL1 receptor reported in the literature, indicating that the hORL1 receptor expressed in this cell line has essentially the same biological characteristics as the native hORL1 receptor, confirming the successful establishment of a stable expression of human opioid receptor-like receptor Cell model.