| Angiogenesis is the most important reason to increasing tumor growth, invasion and metastasis. research shows that abnormal expression of some specific genes promoting angiogenesis, so these antigens expressed on tumor neo-vascular endothelial cells make it become a new therapeutic target of anti-angiogenesis. Develop these agents seem to be capable of suppressing tumor growth specifically without causing systemic toxicity and toxic side effection in non tumor tissues.Integrinαvβ3 isαβheterodimeric receptors expressed on cell surface that mediate divalent cation-dependent cell-cell and cell-matrix adhesion through tightly regulated interactions with ligands. Integrinαvβ3 little expressed in blood vessels during resting state, but expressed rich during inflammation and tumor formation. The abnormal high expression ofαvβ3 promoting tumor angiogenesis and tumor invasion, metastasis. so preventing theαvβ3 integrin, from binding to their ligands such as vitronectin, fibronectin, fibrinogen, laminin, collagen, osteopontin, and others, causes apoptosis in the endothelial cells of newly formed blood vessels, make tumor cells died of insufficient nutrition.Although many research have focused on antagonist of integrinαvβ3, still lacking of peptides or non-peptides angents with high specificity affinity with the integrinαvβ3. Phage display techniques, i.e. the display of libraries of peptides, enzymes, antibodies, and other proteins on the surface of bacteriophages and selection of functional sequences thereof, have undergone a rapid development . Originally used as selection systems to identify peptide epitopes, phage display is today in use for almost any kind of problem that involves the interaction of peptides and proteins with other materials. In this study, we use five short peptides and cultured cells as selected ligands, After three rounds of regular screening or continuous"selective-subtractive-selective"screening, the enriched phage clones were identified by ELISA, The peptides with high affinity to integrinαvβ3 can be obtained through the screening of phage random peptide library with the selected ligands in vitro, which is beneficial to the further studies on the function of integrinαvβ3 in the early diagnosis of cancer and targeting angiogenesis inhibitors specific drug design.The following three experiments were performed in this study.Part I. Use five short peptides as selected ligands to do screening.Use five short peptides as selected ligands what are designed by Crystal Structure of the extracellular segment of integrinαvβ3. After three rounds of screening, phage clones were randomly selected and their affinity was identified by sandwich ELISA. The DNA sequence of positive phage clones was determined, and their speciality was identified by adhesion test. After three rounds of screening, the sequences were obtained, and the consensus sequence was ****HRH and HWR****.Part II. ELISA identify positive phage clones and synthetic peptides.We use two cultured human melanoma cell lines, M2 cells(high expressed integrinαvβ3 ) and C23 cells(no expressed integrinαvβ3). In vitro identify the phage clones and their chemical synthetic peptides binding affinity with the integrinαvβ3. the results show that the phage with the consensus sequences"****HRH and HWR****"and the synthetic peptides"FITC-H L P W H R H,FITC-H W R L H P H"had good binding affinities to M2 cells expressed integrinαvβ3. And this binding can be inhibited by fibronectin.Part III. Subtractive Screening of targeted short binding peptides of integrinαvβ3.To overcome the antigen epitope change when antigen separation and fixation, we use the new subtractive phage display technology to seek short peptides capable of binding integrinαvβ3. After four rounds of binding screening, three rounds of subtractive screening, We finally found 2 of 50 phage clones were identified positive by competitive ELISA, which had comparatively strong binding activity to M2 cells did not to C23 cells. Their sequences were obtained,"H T P P Y I L;M N F N A M L"。...
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