CLONING OF CAPPA-OPIOID RECEPTOR VIA CDNA EXPRESSION

为了回答关于多重阿片类药物的众多问题中的一个 受体，即这些受体是否代表不同的 基因产物，同一基因的不同剪接，或后 产品的跨国改装，是我们追求的目标 目前关于克隆kappa-阿片受体的建议。卡帕- 阿片受体将从任一豚鼠身上克隆出来 小脑或人胎盘文库的表达。 文库中将有丰富的kappa-阿片受体克隆 3套探针：(A)Mu-和 Delta-阿片受体克隆；(B)合成的消减探针 从含有低水平kappa的组织中杂交而来的mRNA- 高水平kappa-阿片受体的阿片受体 受体，即慢性kappa激动剂(U50-488)或拮抗剂 (MR2266)治疗将用于改变kappa-阿片受体 以及(C)寡聚脱氧核苷酸 猪瘟病毒可能的跨膜区V、VI和VII的序列 克隆的8-肾上腺素能受体。杂交的cDNA克隆 使用前两组探头或使用所有三组探头 将在以前没有阿片类药物的真核细胞中表达 受体活性。诱导kappa-阿片受体的克隆 结合活性将被亚克隆到Sp6载体中，用于正义和 反义RNA合成。这样合成的RNA将被 注射入青蛙卵母细胞和kappa激动剂的能力 调节钙离子通道将被用来充实 克隆人的身份。将开发出针对该病毒的抗体 推导出的多肽序列，将用于免疫沉淀 配体-kappa-阿片受体复合体和/或用于抑制 脑膜Kappa-阿片受体结合活性。是这样的 抗体也将用于免疫细胞化学分析 Kappa-阿片受体的分布及其与文献报道的比较 推定的kappa结合位点的分布。这些人的身份 Kappa-阿片受体克隆将进一步得到证实 ~(125)I-B-内啡肽受体复合体的分离和测序 当在Mu和Delta存在的情况下进行标记时- 阿片类配体。猪瘟病毒的基因结构和核苷酸序列 将测定和比较kappa-阿片受体克隆 与Mu-和Delta-阿片受体克隆相同。删除和 将进行核苷酸插入突变研究，以 探讨kappa-阿片受体的结构要求 活动。

In order to answer one of the many questions on the multiple opioid receptors, i.e. whether or not these receptors represent different gene products, different splicing of the same gene, or post- transnational modification of the products, it is the goal of the current proposal to clone for the kappa-opioid receptor. Kappa- opioid receptor will be cloned from either the guinea pig cerebellum or human placenta library by the cDNA expression method. The library will be enriched in kappa-opioid receptor clones with 3 sets of probes: (a) restriction enzyme fragments of the mu- and delta-opioid receptor clones; (b) subtraction probes synthesized from hybridizing mRNAs from tissues containing low level of kappa- opioid receptor from that containing high level of kappa-opioid receptor, i.e. chronic kappa agonist (U50-488) or antagonist (MR2266) treatment will be used to alter the kappa-opioid receptor level and hence mRNA levels; and (c) the oligodeoxynucleotides sequences of the putative transmembrane regions V, VI and VII of the cloned 8-adrenergic receptor. The cDNA clones which hybridize with the first two sets of probes or with all three sets of probes will be expressed in eukaryotes previously devoid of opioid receptor activities. Clones which induced kappa-opioid receptor binding activity will be subcloned into Sp6 vectors for sense and anti-sense RNA synthesis. The RNAs thus synthesized will be injected into frog oocytes and the ability of kappa agonist to regulate the Ca+2 channels will be used to substantiate the identity of the clones. Antibodies will be developed against the deduced peptide sequence and will be used to immunoprecipitate ligand-kappa-opioid receptor complex and/or used to inhibit the kappa-opioid receptor binding activities in brain membranes. Such antibodies will be used also in immunocytochemical analysis of kappa-opioid receptor distribution and compared with the reported distribution of putative kappa binding sites. The identity of the kappa-opioid receptor clone will be substantiated further by the isolation and sequencing of the 125I-B-endorphin-receptor complexes when labelling was carried out in the presence of mu- and delta- opioid ligands. The gene structure and the nucleotide sequence of the kappa-opioid receptor clone will be determined and compared with that of mu- and delta-opioid receptor clones. Deletion and nucleotide insertion mutation studies will be carried out to investigate the structural requirement for kappa-opioid receptor activities.