Purification of opioid receptors and preparation and application of monoclonal antibodies to them

阿片受体的纯化及其单克隆抗体的制备和应用

• 批准号: 63480464
• 负责人: SATOH Masamichi
• 金额: $ 4.29万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-63480464/
• 关键词: Opioid Receptor; Monoclonal Antibody; NG 108-15 Cell; Signal Transduction; オピオイドセレプタ-; オピオイドレセプター; モノクローナル抗体; GTP結合蛋白質

Opioid receptors are classified into three different subtypes, g,&, and K.Opioid receptors are classified into three different subtypes, mu,delta, and kappa. However, little is known of molecular basis of mechanisms of pharmacological actions of opioids through such different subtypes. The present works were focused on the preparation of monoclonal antibodies to opioid receptors and application in order to clarify such molecular mechanisms. Neuroblastoma x glioma hybrid NG108-15 cell membranes were immunized into BALB/C mice for 4 months. The spleen cells were fused with myeloma cells and the culture supernatants in wells containing hybridomas were used for assays in delta-opioid binding experiments. delta-OpR-l showed not only a potent inhibition of delta-opioid agonist binding with a decrease in its Bmax value, but also a blocking of delta-agonist-induced inhibition of adenylate cyclase activity. On the other hand, delta-OpR-3 showed an inhibition of delta-opioid agonist binding with a decrease in its affinity. This antibody was found to stain immunocytochemically delta-opioid receptor expressed in NG108-15 cells. These monoclonal antibodies will be useful for purification of delta-opioid receptors or its expression cloning by immunoscreening methods.

阿片受体分为三个不同的亚型，g、&和K。阿片受体分为三个不同的亚型：u、Delta和kappa。然而，阿片类药物通过不同亚型发挥药理作用的分子基础知之甚少。为了阐明阿片受体的分子机制，本工作主要集中在抗阿片受体单抗的制备和应用方面。神经母细胞瘤×神经胶质瘤杂交瘤NG108-15细胞膜免疫BALB/C小鼠4个月。取脾细胞与骨髓瘤细胞融合，取含杂交瘤细胞的培养上清液进行Delta-阿片结合实验。Delta-OPR-L不仅能有效地抑制Delta-阿片激动剂的结合，降低其Bmax，而且还能阻断Delta-激动剂对腺苷环化酶活性的抑制。另一方面，Delta-OPR-3表现出抑制Delta-阿片激动剂结合的作用，其亲和力降低。该抗体对NG108-15细胞表达的β-阿片受体进行免疫细胞化学染色。这些单抗将为用免疫筛选方法纯化β-阿片受体或克隆其表达奠定基础。

项目成果

植田弘師: "オピオイドレセプタ-機構の分子薬理学" 日本薬理学雑誌. 94. 339-349 (1989)

Hiroshi Ueda：“阿片受体机制的分子药理学”日本药理学杂志 94. 339-349 (1989)。

Ueda,H.,Nozki,M.and Satoh,M.: "Multiple Opioid Receptors and GTP-binding Proteins" Comparative Biochemistry and Physiology,Pergamon Press,oxford, (1990)

Ueda,H.、Nozki,M. 和 Satoh,M.：“多种阿片受体和 GTP 结合蛋白”比较生物化学和生理学，佩加蒙出版社，牛津，（1990 年）

Ueda, H., Nozaki, M. and Satoh, M.: "Multiple opioid receptors and GTP-binding proteins. Comparative Biochemistry and Physiology. in press (1990)" Comparative Biochemistry and Physiology, Pergamon Press, Oxford (1990).

Ueda, H.、Nozaki, M. 和 Satoh, M.：“多种阿片受体和 GTP 结合蛋白。比较生物化学和生理学。出版 (1990)”比较生物化学和生理学，佩加蒙出版社，牛津 (1990)。

Hiroshi,Ueda: "Progress in Endocrinology" Elsevier Science Publishers B.V., 1137-1142 (1988)

Hiroshi,Ueda：“内分泌学进展”Elsevier Science Publishers B.V.，1137-1142 (1988)

植田弘師,佐藤公道: "オピオイドレセプタ-とGTP結合蛋白の再構成" 生物物理. 29. 8-12 (1989)

Hiroshi Ueda、Kimichi Sato：“阿片受体和 GTP 结合蛋白的重建”《生物物理学》29. 8-12 (1989)。