| Summary: | 碩士 === 國立中興大學 === 分子生物學研究所 === 88 === 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.
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