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  • 學位論文

透過噬菌體呈現技術篩選與Integrin α2 I domain 高度專一性之胜肽

Identification of High Affinity Peptides Specific to Integrin α2 I domain by Phage Display Technology

指導教授 : 黃楓婷
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摘要


根據世界衛生組織在2012年的統計報告中顯示,全球男性罹患攝護腺癌的比率佔所有癌症的第二名,且因癌症而死亡的病患人數佔所有癌症的第五名,其中侷限性攝護腺癌 (localized prostate cancer) 的病患在確診五年後的存活率高達百分之八十,而大多數造成死亡的案例是因為攝護腺癌轉移至周遭的骨骼、淋巴腺或是其他器官,所以亟需開發針對轉移型攝護腺癌的診斷平台。Integrin α2β1為type I collagen 主要的受體,且在許多癌症中會過量表現並參與癌症的惡化及轉移的路徑,所以Integrin α2β1為合適的癌症診斷的生物標記,我們的實驗目標為透過噬菌體呈現技術篩選對於integrin α2 I domain具高度專一性的胜肽,並未來將之運用於臨床檢測及分子影像。首先,我們利用C7C phage display library進行biopanning且篩選對於integrin α2 I domain有結合性的環狀胜肽,並且透過phage ELISA binding assay找到兩個與integrin α2 I domain 有結合力的目標胜肽,再利用type I collagen competition assay鑑別目標胜肽與integrin α2 I domain之間的專一性,發現只有C7 胜肽會影響type I collagen與integrin α2 I domain之間的鍵結能力。隨後,利用FITC saturation binding assay測試目標胜肽與integrin α2 I domain的dissociation constant (Kd),發現兩個目標胜肽的Kd落在μM鍵結等級區間,接著將兩個目標胜肽進行in vitro實驗。我們將具有螢光標定的C7或C8胜肽與攝護腺癌細胞PC-3或22Rv1進行反應,透過螢光訊號偵測兩個胜肽結合到攝護腺癌細胞的能力。由實驗結果發現,兩個目標蛋白對於攝護腺癌細胞鍵結強度的差異與其integrin α2β1的表現量成正相關。綜合目前的實驗結果,我們認為C7或C8胜肽在in vitro中具有與integrin α2β1鍵結的能力,而in vivo的實驗正在進行中。

並列摘要


According to WHO cancer statistics in 2012, prostate cancer is the second most common cancer and the fifth leading cause of cancer death in men around the world. Eighty percent of men with the localized prostate cancer are still alive after diagnosis in 5 years, however, at the distant stage of prostate cancer, cancer that have spread to other organs, the survival rate drop dramatically and is the most causes of prostate cancer death. Therefore, it is an urgent need to develop precise and non-invasion diagnostic method for metastatic prostate cancer. The integrin α2β1 is the major type I collagen-binding receptor, and it is overexpressed in many cancer types. The importance of integrin α2β1 involved in prostate cancer metastasis has made it an appropriate biomarker for prostate cancer detection. Hence, in this thesis, the goal was to identify integrin α2β1-binding peptides and ultimately develop as the imaging agent for metastatic prostate cancer diagnosis. First, the recombinant integrin α2 I domain protein was purified and used for selection of high affinity binding peptides with the loop-constrained heptapeptides (C7C) phage library by biopanning. After measurement of the relative binding affinity by phage ELISA binding assay, two integrin α2 I domain-binding phages (C7, C8) were identified. The result of type I collagen competition assay indicated that the C7 phage, but not the C8 phage, could compete with type I collagen to bind to integrin α2 I domain. Moreover, the dissociation constant (Kd) of C7 or C8 peptide to integrin α2 I domain was determined by FITC-peptide saturation binding assay, and the Kd of C7 and C8 peptides were 7 μM and 12 μM, respectively. Furthermore, we used two peptides to target the two prostate cancer cell lines (PC-3 and 22Rv1) that have different integrin α2β1 expression level. By using FITC-labeled C7 peptide or FITC-labeled C8 peptide incubating with these two cell lines and followed by IN Cell Analyzer 2000, we demonstrated that two peptides had greater tendency of binding to PC-3 cells than 22Rv1 cells. In conclusion, C7 or C8 peptide may have potential to be developed as molecular imaging tracer in prostate cancer diagnosis.

參考文獻


Baisi, A., Raveglia, F., & Cioffi, U. (2016). Preoperative Positron Emission Tomography/Computed Tomography in Pulmonary Ground Glass Opacities: A Useful Diagnostic and Staging Tool or Not? Journal of Thoracic and Cardiovascular Surgery, 151(1), 279-280. doi: 10.1016/j.jtcvs.2015.09.033
Bitner, S. E. (2012). False Positives Observed on the Seratec (R) PSA SemiQuant Cassette Test with Condom Lubricants. Journal of Forensic Sciences, 57(6), 1545-1548. doi: 10.1111/j.1556-4029.2012.02141.x
Bonnycastle, L. L., Mehroke, J. S., Rashed, M., Gong, X., & Scott, J. K. (1996). Probing the basis of antibody reactivity with a panel of constrained peptide libraries displayed by filamentous phage. Journal of Molecular Biology, 258(5), 747-762. doi: 10.1006/jmbi.1996.0284
Burdova, A., Bouchal, J., Tavandzis, S., & Kolar, Z. (2014). TMPRSS2-ERG gene fusion in prostate cancer. Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub, 158(4), 502-510. doi: 10.5507/bp.2014.065
Cwirla, S. E., Peters, E. A., Barrett, R. W., & Dower, W. J. (1990). Peptides on phage: a vast library of peptides for identifying ligands. Proc Natl Acad Sci U S A, 87(16), 6378-6382.

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