人C5a受体衍生肽的筛选及其功能的初步研究

发布时间：2018-03-13 06:51

  本文选题：C5a　切入点：CD88　出处：《第三军医大学》2006年硕士论文　论文类型：学位论文

【摘要】： 过敏毒素C5a是补体系统活化产物,也可由一些吞噬性细胞产生,它是炎症反应的重要介质和趋化因子,在急性肺损伤、脓毒血症、脑膜炎、风湿性关节炎、肾小球肾炎等多种疾病的发生发展过程中起重要作用。因此,抑制C5a的生物学活性对于以上炎性疾病的治疗与预防有着重要的意义。 目前的C5a抑制剂大多以C5a受体(CD88)为靶位,通过与C5a竞争性结合CD88而达到抑制C5a生物活性的目的。本研究关注C5a受体上同C5a结合的关键位点,从中筛选既能直接和配体C5a结合,又能拮抗配体C5a生物活性的新型C5a抑制剂,并在此基础上,对其功能作了初步研究。本研究由3个部分组成: 第一部分:C5a结合肽的筛选 利用噬菌体展示肽库技术,首先从噬菌体展示十二肽库筛选阳性克隆,以rhC5a为包被靶分子,将噬菌体展示十二肽库与包被有靶分子的平板共温育,先洗去未结合的噬菌体,然后洗脱与rhC5a特异性结合的噬菌体,将洗脱下来的rhC5a特异性结合的噬菌体进行扩增,再进行下一轮的结合/扩增循环,以富集那些可结合序列。 3次重复以上过程,测序所得阳性克隆,从而得到其氨基酸序列。我们从噬菌体展示十二肽库得到的阳性克隆中随机挑取151克隆进行测序鉴定。 为了更精确地确认C5a与其受体的结合位点,期望找到更精细的拮抗多肽,同时考虑环化肽结构的稳定性,针对噬菌体展示十二肽库的实验结果,本研究又以上述相同的方法,从受限于二硫键环内的噬菌体展示七肽库筛选阳性克隆,同样以rhC5a为包被靶分子,从受限于二硫键环内的噬菌体展示七肽库中我们共挑取138阳性克隆并测序鉴定。 第二部分:C5a受体衍生肽的确定 应用软件Vector NTI Suite 9对以上得到的多肽序列同C5a受体(分别是CD88、C5L2)氨基酸序列进行比对,分析其与C5a受体匹配区域的同源性,从高同源性序列中得到的一系列多肽,我们定义为C5a受体衍生肽。 运用EXPASY工具,分析C5L2的二级结构,结合阳性克隆比对结果,我们分析了
[Abstract]:The allergy toxin C5a is an active product of the complement system and can also be produced by some phagocytic cells. It is an important mediator and chemokine in inflammatory response in acute lung injury, sepsis, meningitis, rheumatoid arthritis, The inhibition of biological activity of C5a plays an important role in the occurrence and development of many diseases such as glomerulonephritis. Therefore, the inhibition of biological activity of C5a plays an important role in the treatment and prevention of the above inflammatory diseases. Most of the current C5a inhibitors target C5a receptor CD88, which can inhibit the biological activity of C5a by competitive binding with C5a. This study focused on the key sites of C5a receptor binding to C5a, and screened the C5a binding directly to the ligand C5a. A novel C5a inhibitor, which antagonizes the biological activity of ligand C5a, has been studied on the basis of which the function of C5a inhibitor has been preliminarily studied. Part one: screening of C5a binding peptides. The phage display peptide library was used to screen the positive clones from the phage display 12 peptide library. The phage display 12 peptide library was incubated with the plate coated with the target molecule, and the unbound phage was first washed out. Then we elucidate the rhC5a specific phage, then amplify the eluted rhC5a specific phage, and then carry on the next cycle of binding / amplification to enrich those binding sequences. The positive clones were sequenced three times and the amino acid sequence was obtained. We randomly selected 151 clones from the phage display 12 peptide library for sequencing. In order to identify the binding site of C5a to its receptor more accurately, and to find more precise antagonistic peptides, and to consider the stability of cyclic peptide structure, we used the same method to display the 12 peptide library by phage display. The positive clones were screened from the phage display heptapeptide library limited by disulfide bond ring, and the rhC5a was also used as the encapsulated target molecule. We selected 138 positive clones from the phage display heptapeptide library limited by disulfide bond ring and identified them by sequencing. Part two: determination of the receptor derivative peptide of: C5a. Vector NTI Suite 9 was used to compare the above peptide sequences with the amino acid sequence of C5a receptor (CD88A C5L2), and to analyze the homology of the matching region with C5a receptor, and a series of polypeptides obtained from high homology sequence. We define C5a receptor-derived peptides. Using EXPASY tool, the secondary structure of C5L2 was analyzed. Combined with the results of positive clone comparison, we analyzed the structure of C5L2.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R341

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