A类清道夫受体胞浆域结合肽的筛选与功能鉴定

发布时间：2018-04-28 22:13

本文选题：动脉粥样硬化 + 泡沫细胞　；参考：《南京医科大学》2006年硕士论文

【摘要】：A类清道夫受体(scavenger receptor-A，SR-A)是一种主要位于巨噬细胞表面的三聚体跨膜糖蛋白，能够识别多种配基，在宿主免疫调节、清除凋亡细胞以及动脉粥样硬化(AS)的发生发展过程中都发挥重要作用。研究表明，SR-A的胞浆域能够调节受体介导的脂质内吞、细胞黏附以及受体在细胞表面表达等过程。去除SR-A胞浆域的受体虽然可以在细胞中表达并能结合乙酰化低密度脂蛋白(AcLDL)，但不能介导AcLDL进入细胞内，细胞不能转化为泡沫细胞。因此，，推测SR-A的胞浆域可能是干预泡沫细胞形成的重要靶点。本研究拟筛选与胞浆域相互作用的小分子多肽，研究这种多肽对SR-A功能的影响，以期研制出能够干预泡沫细胞形成的多肽。在本研究中，首先将SR-A胞浆域编码序列克隆到融合表达载体PGEX-2T中，阳性克隆经IPTG诱导，表达出氨基端融合谷胱甘肽硫转移酶(GST)的SR-A胞浆域蛋白。利用亲和层析法在非变性条件下对表达的目的蛋白进行纯化。经SDS-PAGE电泳及Western blot验证，GST与SR-A胞浆域融合蛋白为可溶性表达，经纯化后目的蛋白纯度大于95％。用纯化的目的蛋白包被谷胱甘肽琼脂糖珠，淘筛噬菌体随机肽库，经过四轮“吸附-洗脱-扩增”亲和筛选，随机挑选50个噬菌体克隆，经噬菌体ELISA法检测到8个噬菌体克隆能特异性结合SR-A的胞浆域，而与GST无交叉反应。抽提阳性克隆DNA并进行序列测定，得到三种多肽序列并命名为：H5、H9、H11。将此
[Abstract]:Class A scavenger receptor scavenger receptor-SR-A-) is a trimer transmembrane glycoprotein mainly located on the surface of macrophages, capable of recognizing multiple ligands in host immune regulation. The removal of apoptotic cells and the development of ASA play an important role. It has been shown that the cytoplasmic domain of SR-A can regulate receptor-mediated lipid endocytosis, cell adhesion and receptor expression on cell surface. The receptors that remove the cytoplasmic domain of SR-A can express in cells and bind to acetylated low density lipoprotein (LDL), but can not mediate AcLDL into the cells, and the cells can not be transformed into foam cells. Therefore, we speculate that the cytoplasmic domain of SR-A may be an important target for the intervention of foam cell formation. The aim of this study was to screen small polypeptides interacting with cytoplasmic domain and to study the effects of the peptides on the function of SR-A in order to develop a polypeptide that could interfere with the formation of foam cells. In this study, the coding sequence of SR-A cytosolic domain was first cloned into the fusion expression vector PGEX-2T, and the positive clone was induced by IPTG to express the SR-A cytosolic domain protein fused with glutathione S-transferase (GST). The expressed target protein was purified by affinity chromatography under non-denaturation conditions. SDS-PAGE electrophoresis and Western blot showed that the fusion protein was soluble expressed in the cytoplasmic domain of SR-A, and the purity of the target protein was higher than 95 after purification. Glutathione agarose beads were coated with purified protein and phage phage random peptide library was screened. After four rounds of affinity screening, 50 phage clones were randomly selected. Eight phage clones were identified by phage ELISA to specifically bind to the cytoplasmic domain of SR-A without cross-reaction with GST. The positive clone DNA was extracted and sequenced. Three kinds of polypeptide sequences were obtained and named as: H5, H9, H11. To change this
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R363

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