ZnF179蛋白相對應抗體之製備及定性

Taipei Medical University Institutional Repository > 醫學科技學院 > 醫學檢驗暨生物技術學系所 > 博碩士論文 > Item 987654321/62467

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題名:	ZnF179蛋白相對應抗體之製備及定性 Generation and characterization of antibodies against zinc finger protein 179 (ZnF179)
作者:	翁郢襌 WENG, YING TSAN
貢獻者:	醫學檢驗暨生物技術學系碩士班楊沂淵
關鍵詞:	鋅指蛋白179;抗體;免疫球蛋白 Y;單鏈抗體 ZnF179;antibodies;IgY;scFv
日期:	2022-07-11
上傳時間:	2023-01-17 15:04:43 (UTC+8)
摘要:	鋅指蛋白179 (ZnF179)，屬於 RING finger 家族中的一員，目前已經證實ZnF179的表現在神經分化的過程扮演重要角色。ZnF179在P19癌細胞中過度表現會導致細胞週期停滯在G0/G1期並啟動神經元細胞分化。進一步的研究表明ZnF179可能通過改變轉錄抑制因子的平衡來影響基因的調控。因此，我們認為開發ZnF179相對應的單株抗體具有其潛力及價值。在本研究中，我們在大腸桿菌中進行ZnF179重組蛋白的表現，並利用鎳離子進行純化再用於雞隻的免疫。我們從雞蛋中純化出多株抗體IgY，並以西方墨點法（Western blot）及酵素結合免疫吸附分析法（ELISA）測試，確認對ZnF179重組蛋白有結合反應。接著我們從雞隻脾臟純化核糖核酸（RNA），並利用噬菌體展示技術（Phage display technology）建構出大小分別為1.0 × 107、1.5 × 107的short linker和long linker的抗體基因庫。經由兩次各4-5輪的篩選（bio-panning），結果顯示兩次的phage-based ELISA都有明顯的上升。我們將52個不同的clone進行抗體重鏈和輕鏈基因序列分析並證實篩選出12株不同的單鏈scFv抗體。在經Western blot及ELISA分析後，證實這些篩選出的scFv抗體對ZnF179重組蛋白具有特異性的結合能力。綜合測試結果，我們選用L1、L2、L4和2S1 scFv抗體，在Western blot和ELISA上進行epitope mapping的分析，推測出抗體和ZnF179重組蛋白的結合形式可能與蛋白的構型（conformation）有所相關。我們也利用Western blot的方式分析抗體對細胞株及小鼠腦組織的結合力，其中L2和L4在Hela、293T細胞株有結合反應但與非人類的小鼠腦組織沒有結合能力。這些scFv單株抗體需要更多的實驗來證實其應用潛力。綜合本篇論文的研究成果，我們認為這些chicken anti-ZnF179 IgY抗體、scFv抗體和scFv抗體基因庫在未來具有潛力可以被應用在針對神經退化相關疾病來發展診斷試劑。 Zinc finger protein 179 (ZnF179) belongs to the RING finger family. Previous studies indicated that the expression of ZnF179 plays an important role in neural differentiation. Over-expression of ZnF179 in P19 carcinoma cells result in the cell arrests in G0/G1 phase and induces the neural cell differentiation. Studies also demonstrated that ZnF179 affects gene regulation by changing the level of transcriptional repressors. Thus, we believed that it has potential to generate the monoclonal antibody against ZnF179. In this study, we expressed recombinant ZnF179 proteins in E. coli and purified the recombinant proteins using Ni2+ for chicken immunization. After purifying polyclonal IgY antibodies from chicken eggs, the IgY antibodies specifically recognized the recombinant ZnF179 proteins on Western blot and ELISA. Subsequently, we isolated the total RNA from chicken spleen, and constructed two scFv antibody libraries by phage display technology, which contained 1.0 × 107 in the short linker and 1.5 × 107 long linker transformants, respectively. After two times of bio-panning (four or five rounds for each), both the phage-based ELISA results indicated that the specific clones were enriched. We selected 52 clones for sequencing and analyzed the light chain and heavy chain gene to classified into 12 different scFv antibodies. These selected scFv antibodies showed specific binding activity to recombinant ZnF179 proteins on Western blot and ELISA analysis. Among of these scFv antibodies, the L1, L2, L4, and 2S1 were used for epitope mapping and speculated that their binding activity might relate to the conformation of ZnF179 protein. We also analyzed the binding activity of selected scFv antibodies to tumor cell lines and mouse brain tissue. Results showed that the L2 and L4 might recognize the ZnF179 in Hela and 293T cells, whereas the mouse brain tissue did not. These scFv antibodies needed to carry out more studies to confirm their application. Taken together, we profoundly believed that these chicken anti-ZnF179 IgY, scFv antibodies and scFv libraries can be applied in developing diagnostic agents for clinical neurodegeneration-related diseases in the future.
描述:	碩士指導教授:楊沂淵委員:楊沂淵委員:劉柯俊委員:柯瓊媛
資料類型:	thesis
顯示於類別:	[醫學檢驗暨生物技術學系所] 博碩士論文

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