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

Identification of High Affinity Peptides Specific to Troponin I, the Ischemic-heart-disease Biomarker, by Phage Display Technology

洪書葶(Shu-Ting Hung)

指導教授 ： 黃楓婷

國立臺灣大學/生命科學院/生化科技學系/碩士(2015年)

https://doi.org/10.6342/NTU.2015.00984

摘要

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。

關鍵字

缺血性心臟病 ； 心臟病指標性蛋白 ； cTnI ； phage display篩選技術 ； 正子造影

並列摘要

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.

並列關鍵字

ischemic heart disease ； cardiac biomarker ； cTnI ； phage display technology ； PET imaging

參考文獻

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