Abstract
Phage display technology, consisting of the expression of target sequences on the surface of phage particles and the subsequent affinity selection, is one of the most powerful tools in studying molecular recognition. The purpose of this study was to select peptide ligands with affinity to 3 hepatocellular carcinoma (HCC) cell lines from phage display random peptide libraries, so that the peptide ligands can be applied to the targeting therapy on hepatoma patients. A 39-mer random peptide library was used for sequential selections against 3 HCC cell lines. Without any amplification of the phages during the selection, 4 clones were obtained. Based on the results of subsequent phage ELISA analysis, one clone exhibiting high affinity to the HCC cell lines was subcloned into pET21c, resulting in the production of His-tagged pep-3 peptide in E coli BL21(DE3). Using pep-3-His peptide as the probe followed by treatment with anti-His-tag monoclonal antibodies, a reactive molecule was visualized. Unfortunately pep-3-His not only reacts with HCC cells but also nasopharygenal carcinoma, hypopharygenal carcinoma, hybridoma and human fibroblast. The result indicates that pep-3 although cannot be used for clinical treatment on hepatoma patients, maybe the pep-3 phage can transfect these cells efficiently since its receptors are abundantly existing on their surfaces. Through the process, a method was developed, which directly detect the peptide binding activity without phage particle. This may serve as a model system to detect the affinity between other selected peptides and their targets. I have also searched for the HCC-binding ligands form phage display C7C random peptide library. Clones with consensus HPQ amino acids were obtained in the control experiment using streptavidin as the selector. Although no obvious consensus sequence could be obtained from 15 randomly picked clones after sequential selection with three HCC cell lines, based on the result of streptavidin panning, it is most likely that the clones recovered from panning against three HCC cell lines sequentially should have some degree of affinity to three cell lines. However more experimental data are needed to prove this.中文摘要
噬菌體展示技術包含了將目標序列展示在噬菌體表面的基因庫構築工作以及後續的篩選過程兩大部分。其最大的特色就是在一個噬菌體顆粒上同時帶有目標基因以及蛋白產物，並且可以透過感染E. coli的過程不斷的複製。利用噬菌體展示系統表現逢機合成的寡核酸序列，就可以製備出具有各種結構的月生 月太 庫，稱為噬菌體逢機月生 月太 庫。藉由噬菌體逢機月生 月太 庫能夠展現各式各樣不同結構的特性，我們對肝癌細胞株進行了一系列的親和篩選，希望能夠找到對其具有專一性的多月生 月太 片段，應用在肝癌的標的療法上。首先以39個逢機胺基酸的月生 月太 庫作為來源，進行連續性親和篩選得到了六個疑似為選殖株的菌落，其中四個帶有插入子（insert）。經過ELISA分析，挑選對三株肝癌細胞株具有較高親和力的一個選殖株，將其插入子以His-tag融合蛋白的方式表現，再藉著anti-His-tag單株抗體的偵測觀察該段月生 月太 的結合情形。結果發現這段月生 月太 是一種細胞表面蛋白的配體，其受體存在於我們所測試的肝癌細胞株、鼻咽癌細胞株、融合瘤細胞株以及人類纖維母細胞上。由於不是肝癌細胞專屬的配體，所以無法應用在肝癌的臨床治療上，但是其結合會結合到所有的細胞上的特色，也許能夠應用在以噬菌體轉化哺乳動物細胞的研究。另一方面，我也使用了7個逢機胺基酸的月生 月太 庫，針對相同的目標進行階段性親和篩選。根據對照組實驗的結果，從streptavidin篩選出來的選殖株，隨機挑選了7株進行DNA定序分析，都具有已知的保守（conserve）胺基酸序列HPQ，顯示階段性篩選相當成功。雖然從肝癌細胞株所篩選到的選殖株，在隨機挑選15株進行DNA定序後，仍然沒有發現明顯的保守序列，但是基於對照組的結果，相信這些選殖株也對肝癌細胞具有某種程度的親和性，然而此一推論仍然需要進一步的實驗來證明。目錄
中文摘要…………………………………………………………………1
英文摘要…………………………………………………………………2
前言………………………………………………………………………3
實驗材料…………………………………………………………………9
實驗方法………………………………………………………………..11
結果與討論……………………………………………………………..31
圖表…………………………………………………………………..…42
參考文獻……………………………………………………………..…56
附錄一………………………………………………………………..…61
附錄二………………………………………………………………..…6
