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研究生:洪書葶
研究生(外文):Shu-Ting Hung
論文名稱:透過Phage Display技術篩選與缺血性心臟病指標蛋白Troponin I高度專一性之胜肽
論文名稱(外文):Identification of High Affinity Peptides Specific to Troponin I, the Ischemic-heart-disease Biomarker, by Phage Display Technology
指導教授:黃楓婷黃楓婷引用關係
指導教授(外文):Feng-Ting Huang
口試委員:周綠蘋許益瑞廖憶純
口試日期:2015-07-16
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科技學系
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:79
中文關鍵詞:缺血性心臟病心臟病指標性蛋白cTnIphage display篩選技術正子造影
外文關鍵詞:ischemic heart diseasecardiac biomarkercTnIphage display technologyPET imaging
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急性心肌梗塞為缺血性心臟病中致死率極高的一類。現今臨床上有許多用於偵測急性心肌梗塞的指標性蛋白,包含cardiac troponin I (cTnI)。cTnI具有極高的心肌特異性,當心肌受損時釋放至血液中,並且在至少七天內仍然可以偵測到血液中的cTnI。我們的實驗目標為透過phage display技術篩選對cTnI具有高度專一性的胜肽,並將之運用於臨床檢測及分子影像。
首先,我們利用兩種不同的phage display library進行biopanning,篩選與cTnI有結合性的環狀和直鏈胜肽,並且透過ELISA找到九個與cTnI有結合力的目標胜肽。因為N端為心肌特有的部份,因此我們也利用cTnI的N端進行biopanning,並找到了目標胜肽NC4-1。由實驗結果發現,十個目標胜肽都會結合到cTnI的中間區域,並且有三個也會結合到cTnI的N端。此外我們也發現,含有血清的細胞培養液並不會干擾目標胜肽與cTnI的結合力。
根據實驗結果,比較每個目標胜肽與cTnI的dissociation constant (Kd),我們選擇與cTnI具有較高結合性並且能與N端結合的NC4-1進行in vitro和in vivo實驗。我們將具有螢光標定的NC4-1胜肽與大鼠心肌母細胞株 H9c2 (2-1) 反應,透過螢光訊號偵測NC4-1胜肽結合到心肌細胞的能力。由實驗結果發現,NC4-1胜肽會聚集於細胞質中,而cTnI也存在於細胞質中。進一步利用分子影像的技術,觀察NC4-1是否能偵測缺血性心臟病大鼠model中心肌受損的區域。由PET影像結果發現, 68Ga標定的NC4-1 phage可結合至心肌受損的區域,並且此區域與SPECT影像中正常的心肌區域互補。綜合目前的實驗結果,我們認為NC4-1胜肽具有應用於急性心肌梗塞臨床偵測的潛力。


Acute myocardial infarction (AMI) is the most common type of ischemic heart disease. Many clinical biomarkers have been used for detection AMI including cardiac troponin I (cTnI). cTnI is a cardiac-specific isoform of troponin I. Plasma cTnI increases rapidly after cardiomyocyte damage and remains detectable after days. Our goal is to identify high affinity peptides specific to cTnI and to apply in serum detection and molecular imaging for clinical use.
First, we used two different phage libraries to select cyclic 7-mer and linear 12-mer peptides by biopanning experiments. We found nine candidate peptides with high binding affinities to purified recombinant human cTnI after checking by ELISA binding assay. Since the N-terminal region is unique to the cardiac isoform of cTnI, we also performed biopanning against this region of cTnI and identified NC4-1. Next, we found all candidates bound to the central region, and three candidates bound to the N-terminal region. In addition, we found that the binding affinities of candidates to cTnI were not affected by culture media with fetal bovine serum.
For further analysis, the apparent dissociation constants of candidate phage clones and their corresponding synthesized peptides were measured. According to our results, we indicated that candidate NC4-1 had better binding affinity to cTnI. Moreover, candidate NC4-1 could target the N-terminal region of cTnI. We then used candidate NC4-1 to target cTnI in the rat heart myoblast cell line and ischemic-heart-failure rats. By using FITC-labeled NC4-1 peptide, we demonstrated that NC4-1 peptide could target to the cytoplasm of H9c2 (2-1) cells, and that was corresponding to the localization of cTnI studied by ICC using cTnI antibody. For in vivo study, PET imaging was performed using 68Ga-phage NC4-1 as the tracer to target cTnI in ischemic-heart-failure rats. Comparing the region identified as normal cardiac muscle by SPECT imaging using 99mTc sestamibi, we found that candidate NC4-1 could target to damage sites of cardiac muscles. In conclusion, NC4-1 peptide may have the potential to be developed as a diagnostic molecular imaging tracer in AMI detection.


謝辭 i
中文摘要 ii
Abstract iii
Table of Contents v
Chapter 1 Introduction 1
1.1 Acute myocardial infarction (AMI) 1
1.2 Cardiac troponin I 2
1.3 Current approach for detection cTnI 4
1.4 Phage display technology 5
1.5 Molecular imaging 7
1.6 Research Purpose 8
Chapter 2 Materials and methods 10
2.1 Expression and purification of recombinant human cTnI protein 10
2.2 Biopanning 11
2.2.1 Biopanning for cTnI 11
2.2.2 Biopanning for the N-terminal (1-35 aa) region of cTnI 12
2.3 Phage Titering 14
2.4 Extraction of phage single-stranded DNA 14
2.5 Phage amplification 16
2.6 Phage ELISA (enzyme-linked immunosorbent assay) binding assay 17
2.7 Cloning and expression of different fragments of cTnI 17
2.8 Measurement the dissociation constant between phage and cTnI protein 19
2.9 Effect of culture medium and serum on binding affinity by ELISAs 19
2.10 FITC-peptide saturation binding assay 20
2.11 Surface plasmon resonance 21
2.12 Phage-antibody sandwich ELISAs 22
2.13 in vitro experiment 22
2.13.1 Cell culture 22
2.13.2 Hypoxia treatment 23
2.13.3 Immunocytochemistry (ICC) 23
2.13.4 Peptide binding in vitro 24
2.14 Western Blotting 24
2.14.1 Whole cell lysate preparation 24
2.14.2 Gel electrophoresis 25
2.14.3 Transfer 25
2.14.4 Immunoblotting 25
2.15 in vivo experiment 26
2.15.1 Ischemic-heart rat model 26
2.15.2 Immunohistochemistry (IHC) 26
Chapter 3 Results 28
3.1 Expression of recombinant human cTnI 28
3.2 Identification of cTnI binding phage through biopanning 28
3.3 Characterization of the selected phage clones by the ELISA binding assay …………………………………………………………………………….29
3.4 Identification of phage clones targeting the N-terminal region of cTnI through biopanning 30
3.5 The binding site of candidate phage clones on cTnI 31
3.6 Measurement of the apparent dissociation constant of candidate phage clones …………………………………………………………………………….32
3.7 Effects of culture media and serum on the binding affinity of candidate phage clones to cTnI 32
3.8 Evaluation the binding affinity of selected peptides to cTnI 33
3.9 Detection of cTnI by phage-antibody sandwich ELISA 34
3.10 Binding of peptide NC4-1 to the rat heart myoblast cell line H9c2 (2-1) …………………………………………………………………………….34
3.11 Molecular imaging using phage NC4-1 as tracer 35
3.12 Ex vivo analysis of phage NC4-1 targeting sites on ischemic-rat-heart 36
Chapter 4 Discussion 38
4.1 Biopanning procedure 38
4.2 Binding sites of selected phage clones on cTnI 39
4.3 Comparison between the peptide and the corresponding phage clone 39
4.4 Alternative approach to detect cTnI using peptides 40
4.5 Targeting cTnI in vitro 41
Chapter 5 Summary and future prospects 42
Chapter 6 Reference list 43
Figures and tables 46
Figure 1. CBR staining of purified recombinant His-tag human cTnI 47
Figure 2. Characterization of the human cTnI-binding-phage clones using a titration ELISA binding assay 48
Figure 3. Characterization of the N-terminal binding phage clone NC4-1 using a titration ELISA binding assay 50
Figure 4. CBR staining of the purified recombinant His-tag C-terminal and central fragments of human cTnI 51
Figure 5. ELISA binding assay of candidate phage clones to different fragment of recombinant human cTnI 52
Figure 6. Apparent dissociation binding constant (Kd) of candidate phage clones measured by indirect ELISA 53
Figure 7. Effects of culture media and serum on the binding affinity of candidate phage clones to recombinant human cTnI 56
Figure 8. The binding affinity of candidate peptides to cTnI by the saturation binding assay using FITC-labeled peptides 57
Figure 9. The surface plasmon resonance (SPR) analysis of the peptide FC2-2, FL6-5 and NC4-1 60
Figure 10. Capture of cTnI by phage-antibody sandwich ELISA with phage NC4-1 62
Figure 11. Confirm the localization of cTnI in the rat heart myoblast cell line H9c2 (2-1) under normal or hypoxia condition 63
Figure 12. The protein level of cTnI in the rat heart myoblast cell line H9c2 (2-1) under normal or hypoxia condition 64
Figure 13. Binding of peptide NC4-1 to the rat heart myoblast cell line H9c2 (2-1) under normal or hypoxia condition 65
Figure 14. PET and SPECT imaging of the ischemic-heart-failure rat 66
Figure 15. Autoradiography (ARG) and immunohistochemistry (IHC) images of ischemic rat hearts 67
Table 1. Enrichments obtained during biopanning with the C7C phage display library against purified His-tag human cTnI 68
Table 2. Amino acid sequences and frequency of the selected phage clones using the C7C phage display library against purified His-tag human cTnI 69
Table 3. Enrichments obtained during biopanning with the L12 phage display library against purified His-tag human cTnI 70
Table 4. Amino acid sequences and frequency of the selected phage clones using the L12 phage display library against purified His-tag human cTnI 71
Table 5. Enrichments obtained during biopanning with the C7C phage display library against the synthesized N-terminal region of human cTnI (1-35 aa) 72
Table 6. Amino acid sequences and frequency of the selected phage clones using the C7C phage display library against the synthesized N-terminal region of human cTnI (1-35 aa) 73
Table 7. Apparent dissociation binding constant (Kd) of candidate phage clones as measured by indirect ELISA 74
Table 8. Apparent dissociation binding constant (Kd) of candidate peptides as measured by the saturation binding assay using FITC-labeled peptides 75
Appendixes 76
A.Details of primers 77
B.PCR conditions 77
C.Antibodies 78
D.Buffer 79




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