人CD59活性位点短肽封条的抗肿瘤效应的研究

发布时间：2018-01-09 07:18

本文关键词：人CD59活性位点短肽封条的抗肿瘤效应的研究　出处：《青岛大学》2007年硕士论文　论文类型：学位论文

【摘要】： 目的本研究旨在通过噬菌体展示技术寻找与肿瘤逃逸相关的CD59活性位点，研制特异性针对CD59活性位点的短肽封条，观测其封闭或干扰效应，为肿瘤的靶向免疫治疗开辟一条新途径。方法采用噬菌体随机十二肽库展示技术对Hela细胞裂解蛋白及层析纯化后的CHO细胞的裂解蛋白进行5轮亲和淘洗筛选；从筛选后的噬菌体库中分别随机挑选15／16个噬菌体克隆进行测序，并对随机挑选的噬菌体克隆与野生型M13噬菌体进行竞争结合实验；经序列分析找出共享序列并推断其相应的氨基酸位点，，同时与CD59活性区域进行分析比对，找出人CD59分子的活性位点，并合成短肽封条，观测其封闭效应。结果经过5轮全细胞筛选后，两种噬菌体库的回收量均可达到10~7～10~8数量级，且竞争结合分析及细胞特异性结合分析显示筛选后的噬菌体克隆及噬菌体库对两种细胞蛋白均具有明显的特异性结合效应。通过测序结果推断出Hela细胞表面CD59的多肽序列(—ACHWPWCHGC—)与CHO细胞表面CD59的多肽序列(—ACHWWHAWTC—，—ACWWFHPHLC—)，并利用高同源性的Hela细胞的CD59短肽序列为模型合成高特异性的短肽封条。结论利用噬菌体肽库对两种细胞进行全细胞蛋白筛选得到了与CD59特异性结合的短肽序列，该短肽序列可能是位于肿瘤细胞表面CD59的配体蛋白。经活性检测证实该短肽封条对人CD59有明显的封闭作用。
[Abstract]:Objective The aim of this study was to find CD59 active site related to tumor escape through phage display technique , and to develop a short peptide seal specific for CD59 active site , and to observe its blocking or interference effects , which opened a new way for targeted immunotherapy of tumors . Methods A phage random twelve peptide library was used to screen the lysate of Hela cell lysate and purified CHO cells . 15 / 16 phage clones were randomly selected from the screened phage library for sequencing , and the random selection phage clones were randomly selected to compete with the wild type 13 phage . The sequence analysis was used to identify the corresponding amino acid sites and to identify the active sites of CD59 molecules and to synthesize short peptide seals to observe the blocking effect . Results After five rounds of whole cell screening , the recovery of two phage libraries could be up to 10 ~ 7 ~ 10 ~ 8 orders of magnitude , and the competitive binding analysis and cell - specific binding assay showed that the phage clones and phage libraries had obvious specific binding effect on both cell proteins . The polypeptide sequences of CD59 ( ACHWPW CHGC - ) and CD59 on the surface of CHO cells were deduced by sequencing . The high - specific short peptide seal was synthesized by using the CD59 short peptide sequence of Hela cells with high homology . Conclusion The short peptide sequence which specifically binds CD59 is obtained by using phage peptide library to screen two kinds of cells . The short peptide sequence may be a ligand protein located on the surface of tumor cell CD59 . The activity test proves that the short peptide seal has obvious blocking effect on human CD59 .

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R392;R730.5

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