CLONING AND EXPRESSION OF OPIOID RECEPTOR GENES

阿片肽和药物在中枢和中枢神经系统引起多种作用 遍及全身。几种不同的阿片受体已经被 描述了生物化学和药理学，但编码基因 他们仍然不为人所知。我们建议鉴定和克隆阿片受体 并将每一种基因都导入哺乳动物细胞。因为我们已经确定了 并对人神经母细胞瘤细胞系(SH-SY5Y)进行了全面鉴定 即表达丰富的Mu阿片受体，研究将重点放在Mu 第一阶段的阿片受体基因分离策略包括 1.使用由以下多肽片段衍生的寡核苷酸探针 MU受体蛋白；2.cdna的直接哺乳动物表达克隆。 来自SH-SY5Y细胞的文库，或者，表达克隆 显微注射受体信使核糖核酸的蛙卵母细胞3.基因的筛选 具有来自任何已知已知基因的探针的密切相关基因文库 阿片受体基因。扩大搜索范围将集中在主要的阿片类药物上 受体分类：Mu、Delta和Kappa，以及它们可能的亚型。 对不同阿片受体基因数量及其相互关系的认识 分子结构和稳定转基因细胞的可用性 将允许开发明确的阿片类药物检测方法 在每一种受体亚型上的作用，从而提供强大的 开发高度特异性新药的工具。这些大体 目标将在该项目的第二阶段得到解决。

Opioid peptides and drugs cause multiple effects in the CNS and throughout the entire body. Several distinct opioid receptors have been described biochemically and pharmacologically, but the genes encoding them remain unknown. We propose to identify and clone the opioid receptor genes and transfect each into mammalian cells. Because we have identified and thoroughly characterized a human neuroblastoma cell line (SH-SY5Y) that expresses abundant mu opioid receptors, studies will focus on the mu opioid receptor during phase I. The strategies for gene isolation include 1. the use of oligonucleotide probes derived from peptide fragments of the mu receptor protein; 2. direct mammalian expression cloning of cDNA libraries from SH-SY5Y cells, or alternatively, expression cloning in frog oocytes micro-injected with receptor mRNA 3. screening of cDNA libraries for closely related genes with probes derived from any known opioid receptor gene. The extended search will focus on the main opioid receptor classes, mu, delta and kappa, and their putative subtypes. Knowledge of the number of distinct opioid receptor genes and their molecular structure, and the availability of stably transfected cell lines, will permit the development of unequivocal assays of opioid effects at each of the receptor subtypes, and thereby, provide a powerful tool for the development of highly specific novel drugs. These general objectives will be addressed in phase II of this project.