CLONING OF A PUTATIVE NON OPIOID BINDING PROTEIN OF DNA

当前提案的目的是克隆非阿片类药物结合蛋白。 内源性阿片肽强啡肽 A-(1-17) 及其类似物强啡肽 A-(2-17) 对 kappa 阿片受体具有高亲和力，但大量证据表明它们也与非阿片蛋白结合。 这种蛋白质的鉴定受到强啡肽的几个特征的阻碍，包括其与生物组织的高度非特异性结合、其对蛋白水解降解的敏感性以及缺乏对其一级结构的了解。 这里提出了两种互补的方法来规避这些问题，两种方法都应用于强啡肽A-(2-17)，它不与阿片受体结合，并且是假定的非阿片蛋白的更具选择性的配体。 首先，酵母双杂交系统将用于识别与 DynA-(2-17) 相互作用的任何分子。 其次，表达克隆和同胞选择将用于鉴定结合 DynA-(2-17) 的克隆。 DynA-(1-17) 及其类似物 dynA-(2-17) 在大脑中没有镇痛作用，但可以增强吗啡的镇痛作用并抑制吗啡耐受/依赖性动物的戒断症状。 因此，它在提高长期需要吗啡的患者的疗效以及治疗阿片类药物成瘾方面具有巨大的临床前景。鉴定介导这些效应的结合蛋白对于这些进展至关重要。

The purpose of the current proposal is to clone a non-opioid binding protein. The endogenous opioid peptide dynorphinA-(1-17) and its analog dynorphinA-(2-17) have high affinity for kappa opioid receptors but considerable evidence indicates that they also bind to a non-opioid protein. Identification of this protein has been hindered by several features of dynorphin, including its high non-specific binding to biological tissue, its sensitivity to proteolytic degradation and a lack of knowledge of its primary structure. Two complementary approaches are proposed here to circumvent these problems, both being applied to dynorphinA-(2-17), which does not bind to opioid receptors and would be a more selective ligand for the putative non-opioid protein. First, the yeast two-hybrid system will be used to identify any molecules interacting with DynA-(2-17). Second, expression cloning and sib selection will be used to identify clones that bind DynA-(2-17). DynA-(1-17) and its analog dynA- (2-17) are not analgesic in the brain but can enhance morphine antinociception and suppress withdrawal symptoms in morphine tolerant/dependent animals. Thus, it has great clinical promise in improving the efficacy of morphine in patients who require the drug chronically, as well as a treatment for opioid addiction. Identification of the binding protein mediating these effects is critical to these advances.