臺灣博碩士論文加值系統

Integrin family是一種在各類癌症中高表現的蛋白質族群。其中Integrin α2更是被證明了與癌症發展的高度相關性。在本實驗室的先前研究中已表明了，當抗體與癌細胞表面的Integrin α2結合後促使細胞提高Capase-9 的活性而導致細胞凋亡。根據上述的結果，我們利用生物資訊分析的軟體分析Integrin α2蛋白質序列並同時運用不同分析面向，找出蛋白質片段Peptide-2可能是一個有效的抗原決定位。我們利用這段可能是抗原決定位的蛋白質片段，成功誘發小鼠免疫反應，並得到會專一性辨認細胞表面Integrin α2之抗體。同時發現抗體具有毒殺胃癌細胞的能力。本研究結果顯示我們成功建立一套抗原決定位的分析系統，以及製造出具有辨識Integrin α2以及抑制胃癌細胞生長的抗體。

Integrin family is a well-known cancer-overexpression-protein family. Integrin α2 (ITGA2), a member of this family, has been proved to be a tumor antigen in many cancers and to promote migration of cancer cells, including gastric cancer. Our previous research showed that ITGA2 had another functional pathway. According to the preliminary data, using the anti-ITGA2 antibody to bind to gastric cancer cell line can induce cell apoptosis. Our data shows that once antibody binds to ITGA2 on surface of cancer cell will induce Capase-9 to promote cell death. Therefore, we decided to clarify which domain of ITGA2 can be bound by antibody and lead to cell apoptosis. We used several computational tools to help predicting relevant B cell epitopes within ITGA2. Our prediction showed that Peptide-2 might be an efficacious binding site of antibody on gastric cancer cells. To verify the prediction of the binding epitope, we immunized BALB/c with ITGA2 peptide fragment and plasmid, including Peptide-1, Peptide-2, pITGA21-500, and pITGA2501-1181 to mouse. After immunization, we used mouse anti-serum to treat on gastric cancer cell line. According to cell vibration rate, we found out that groups immunized with Peptide-2 has better response than Peptide-1, pITGA21-500 plasmid, and pITGA2501-1181 plasmid. In conclusion, we find out and experimentally conform that Peptide-2 is a potential epitope and can inhibit gastric cancer cell growth.

中文摘要 I
ABSTRACT II
致謝 IV
CONTENTS V
CHAPTER1. INTRODUCTION 1
CHAPTER2. MATERIAL AND METHODS 5
2.1 Cells and culture conditions 5
2.2 Integrin α2 3D structure model 5
2.3 B Cell Epitope Prediction 6
2.4 Prediction of Intrinsically Unstructured/Disordered Regions (IURs) and Potential Binding Regions in integrin α2 6
2.5 Integrin α2 Protein Surface Hydrophobic Molecular Modeling 7
2.6 Protein Post-Translation Modification Modeling 7
2.7 Peptide Synthesis 7
2.8 Preparation of the Peptide-LPPC (Lipo-PEI-PEG complex) complex and DNA-LPPC complex 8
2.9 Immunization and Titer Boost 8
2.10 Detection of Produced Antisera against the integrin α2133-167 and integrin α2407-441 9
2.11 Detection of Produced Antisera against the integrin α2 on gastric cancer cell surface 9
2.12 Assay of cell proliferation rate 10
2.13 Antibody sequence analysis 10
2.14 Bioinformatics Data Base - The Cancer Genome Atlas 11
2.15 Statistical Analysis 11
CHAPTER3. RESULTS 12
3.1 ITGA2 gene expression level and cellular function 12
3.2 In silico analysis of integrin α2 and identification of potential B-cell epitopes 13
3.3 Protein modeling of the integrin α2 13
3.4 Predicting ITGA2133-167 and ITGA2407-441 as potential B-cell epitopes 14
3.5 Antisera specifically response to the immunizing peptide fragment 15
3.6 The ability of antisera induced by different fragment to detect gastric cancer cell surface integrin α2 16
3.7 The effects of antisera on cell viability in gastric cancer cell line 16
3.8 Cross-reacting on mouse B-lymphoblast 17
3.9 Protein sequencing to get antibody sequence in the antisera 17
CHAPTER4. DISCUSSION 18
TABLE 1. ITGA2 AND ERBB2(HER2) GENE EXPRESSION LEVEL IN GASTRIC CANCER CLINICAL SAMPLES AND PARA-CANCER TISSUES. 21
TABLE 2. POST-TRANSLATION MODIFICATION PREDICTION ON EUKARYOTIC LINEAR MOTIF (ELM) 21
FIGURE 1. ITGA2 GENE EXPRESSION LEVEL ON GASTRIC CANCER. 22
FIGURE 2. PROTEIN INTERACTION NETWORK ABOUT ITGA2. 23
FIGURE 3. PREDICTION SCORES FOR LINEAR B CELL EPITOPES, INTRINSICALLY UNSTRUCTURED/DISORDERED REGIONS AND PROTEIN-PROTEIN INTERACTION REGIONS. 24
FIGURE 4. THREE-DIMENSIONAL STRUCTURE PREDICTION ON ROBETTA SEVER. 25
FIGURE 5. MODIFICATIONS AND HYDROPHOBIC PHASE ON INTEGRIN Α2. 26
FIGURE 6. THE CHOSEN PEPTIDES ON INTEGRIN Α2. 27
FIGURE 7. IMMUNIZED TITER AND ANTIBODY BINDING ABILITY TO ITGA2133-167 AND TO ITGA2407-441. 28
FIGURE 8. USING PURIFIED ANTIBODY TO BIND TO CELL SURFACE INTEGRIN Α2. 29
FIGURE 9. EFFECTS OF INDUCED ANTIBODY ON CELL PROLIFERATION IN GASTRIC CANCER CELL LINE 30
FIGURE 10. ANTI-ITGA2407-441 CAN BIND TO MOUSE B-LYMPHOBLAST 31
FIGURE 11. HUMAN AND MOUSE ITGA2407-441 SEQUENCE BLAST 31
FIGURE 12. ANTIBODY PURIFIED 32
FIGURE 13. MOUSE IG GAMMA-1 C REGION ANTIBODY SEQUENCE ALIGNMENT 33
FIGURE 14. MOUSE IG KAPPA-1 C REGION ANTIBODY SEQUENCE ALIGNMENT 34
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