CLONING AND EXPRESSION OF OPIOID RECEPTOR GENES

阿片肽和药物在CNS中引起多种作用， 遍布全身几种不同的阿片受体已经被 在生物化学和生物化学上进行了描述，但是编码 他们仍然是未知的。我们建议鉴定和克隆阿片受体 基因并将其导入哺乳动物细胞。因为我们发现 并彻底表征了人神经母细胞瘤细胞系（SH-SY 5 Y） 表达丰富的μ阿片受体，研究将集中在μ 阿片受体I期。基因分离的策略包括 1.衍生自以下肽片段的寡核苷酸探针的用途： mu受体蛋白; 2. cDNA的直接哺乳动物表达克隆 来自SH-SY 5 Y细胞的文库，或者可替代地，在 显微注射受体mRNA的蛙卵母细胞3.筛选cDNA 与来自任何已知的探针的密切相关的基因的文库 阿片受体基因扩展搜索将集中在主要的阿片类药物上 受体类别，μ、δ和κ，以及它们推定的亚型。 了解不同阿片受体基因的数量及其 分子结构和稳定转染细胞的可用性 线，将允许开发阿片类药物的明确测定 在每个受体亚型的作用，从而，提供了一个强大的 开发高度特异性新药的工具。这些一般 这些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.