daintainaif1酵母双杂交诱饵表达载体的构建及初步鉴定word格式论文.docxVIP

摘要酵母双杂交系统是一种直接在真核细胞内检测蛋白相互作用的方法，具有灵敏 度高和特异性好的优点。自 1989 年由Fields等提出并初步建立以来，得到了不断的 完善和改进，在细胞生物学，肿瘤学，蛋白质组学等诸多领域有着广泛的应用。 Daintain是 1994 年Chen ZW等从猪小肠粘膜中分离纯化出的含 146 个氨基酸的生物 活性肽，与Utans等鉴定的异体移植炎症因子(AIF-1)的序列高度相似，合称 为 Daintain/AIF-1。1997 年，Chen ZW等继续报道了Daintain对大鼠胰腺β细胞分泌胰 岛素具有双向影响，以及在前驱糖尿病进程中，在胰腺中超量表达Daintain的巨噬 细胞浸润胰岛，显示Daintain/AIF-1 与胰岛素依赖型糖尿病的发病机理有关。目前 已经证实，Daintain/AIF-1 广泛存在于脾脏和胸腺等免疫器官以及脑、肝脏、肺， 胎盘和睾丸等非免疫器官中，是一个具有多种生理功能的活性蛋白，参与人体Ⅰ型 糖尿病、胰腺炎、脑神经炎等免疫失调疾病反应。基于此，我们拟用酵母双杂交GAL4 系统，构建BD-Daintain/AIF-1 融合蛋白即诱饵蛋白，以期从人胰腺cDNA文库中钓取 可与之相互作用的蛋白的基因，为进一步研究Daintain/AIF-1 对胰岛素分泌调节提 供实验依据。我们构建了 Daintain/AIF-1 的酵母双杂交诱饵表达载体 pGBKT7-AIF-1，经测序 鉴定正确后转化入 AH109 酵母，检测证明单独的 BD-Daintain/AIF-1 诱饵融合蛋白 不能激活报告基因的转录；对酵母细胞的生长也没有毒性。因此，我们构建的诱饵 蛋白表达载体 pGBKT7-AIF-1 符合筛选 cDNA 文库的要求，解决了本实验的关键性问 题，可以进行 Daintain/AIF-1 的相互作用蛋白的筛选，为通过酵母双杂交研究 Daintain/AIF-1 的功能及作用机制奠定了基础。关键词：Daintain/AIF-1，酵母双杂交系统，载体构建AbstractThe yeast two-hybrid system is a powerful and sensitive molecular genetic approach for studying protein-protein interactions in vivo. Over the seventeen or so years since the first report of the system by Fields and Song, it has been modified in numerous ways and has been widely adapted for the study of cell biology, oncology, proteomics and so on. Daintain was a bioactive macrophage factor discovered by Chen ZW in 1994 and was first isolated from porcine intestine. It is a 146-aa residue polypeptide and the amino acid sequence is highly identical to that of rat and human allograft inflammatory factor-1(AIF-1), so we named it Daintain/AIF-1. In 1997 Chen ZW reported that Daintain/AIF-1 exerted a dual influence on insulin secretion in vivo at different peptide concentrations and a particularly dense accumulation of Daintain/AIF-1-immunoreactive macrophages was observed in the insulitis affecting the pancreatic islets of prediabetic BB rats, suggested that Daintain/AIF1 may have a role in connection with the pathogenesis of insulin-dependent diabetes mellitus. Daintain/AIF-1 has been detected in the red pulp of s