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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 湯志永 | zh_TW |
dc.contributor.advisor | Chih-Yung Tang | en |
dc.contributor.author | 羅祥 | zh_TW |
dc.contributor.author | Hsiang Lo | en |
dc.date.accessioned | 2021-07-11T15:40:31Z | - |
dc.date.available | 2024-02-28 | - |
dc.date.copyright | 2018-10-11 | - |
dc.date.issued | 2018 | - |
dc.date.submitted | 2002-01-01 | - |
dc.identifier.citation | Aggarwal, S. K. & MacKinnon, R. (1996) Contribution of the S4 segment to gating charge in the Shaker K+ channel. Neuron. 16:1169-1177.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/79059 | - |
dc.description.abstract | 人類基因KCND3 encode出電位調控鉀離子通道Kv4.3,Kv4.3屬於Shal-type family (Kv4 family)次家族成員之一,廣泛表現在整個大腦、心臟及平滑肌當中,並且已有研究指出當病人KCND3基因發生突變時會導致SCA19/22脊髓小腦萎縮症;顯性體染色體遺傳性疾病,目前文獻指出當位於小腦的Purkinje cell中發生突變的Kv4.3 α subunit,會dominant negative影響wild type Kv4.3α subunit由ER送至細胞膜上表現;也會與wild type Kv4.3 α subunit進行聚合,並且影響通道的功能,但是對於Purkinje cell死亡機制目前尚不清楚,為了對Kv4.3有更進一步的認識,我們利用酵母雙雜交系統尋找與Kv4.3交互作用之蛋白質,希望能透過了解這些蛋白質在細胞中已知參與的機制、訊息傳導路徑,進一步推測對Kv4.3的調控性質。在對於大鼠腦部cDNA library進行篩選後,我們共發現了318個clonies可能與Kv4.3通道蛋白有交互作用,在經過進一步的序列分析和扣除胺基酸序列有frame shift後,剩下12個可能與Kv4.3通道蛋白有交互作用的蛋白質,我們從中挑選出來7個作為研究目標,進一步以實驗證明與Kv4.3通道蛋白交互作用的能力。首先我利用酵母雙雜交實驗 (包含X-gal測試分析與Leucine需求分析實驗)以釐清Kv4.3和其可能相互作用蛋白質之相關性。在酵母雙雜交實驗中,這12個蛋白質都能在進行X-gal測試分析實驗使菌落由白變藍,並且在缺乏白胺酸的培養皿上生長。我們進一步以GST pull down與Co-IP實驗之方式驗證,發現各有5個得到正反應。針對本篇研究所篩選到的蛋白質,我們除了以不同的生化實驗證明他們的交互作用外,我們期望將來能夠以更多生化實驗及電生理對這些蛋白質與Kv4.3之間的交互作用更深入的探討與分析,以了解Kv4.3在細胞內的各種生化機制。 | zh_TW |
dc.description.abstract | Human gene KCND3 encode voltage-gated potassium channel Kv4.3. Kv4.3 highly express in brain, heart, and smooth muscle, which belong to Shal-type family (Kv4 family). Spinocerebellar ataxia type 19/22 (SCA19/22) is a dominantly inherited neurodegenerative, clinically heterogeneous disorder caused by mutations in KCND3 and Purkinje cell loss in the cerebellum consequently. SCA19/22mutant Kv4.3 subunits exerted dominant negative effects on WT Kv4.3 either in endoplasmic reticulum (ER)-Golgi membrane trafficking or reduced Kv4.3 channel functioning.In order to understand further about Kv4.3, we applied C-termius and N-terminus of Kv4.3 as the bait for yeast two-hybrid screening to search for novel Kv4.3 interacting proteins.
318 prey clonies were identified after screening a rat brain cDNA library. By X-gal assays, leucine requirement tests, yeast plasmid extract, colony PCR, sequencing and eliminating the clones with frame shit, we have obtained 12 potential candidate proteins. We chose 7 candidates for further characterization. GST pull down assay and Co-IP assay were performed to reconfirm the interaction between Kv4.3 channels and candidates. We found the five of the seven candidates have positive reaction in GST pull down assay; and found the five of the seven candidates have positive reaction in Co-IP assay. In this study, we found novel candidate proteins interacting with Kv4.3, and we performed experiments to prove it. We hope to better understand about physiological mechanism of Kv4.3 potassium channel with further biochemical experiment and electrophysiology experiments. | en |
dc.description.provenance | Made available in DSpace on 2021-07-11T15:40:31Z (GMT). No. of bitstreams: 1 ntu-107-R04441012-1.pdf: 1634698 bytes, checksum: 29daf57d1fc94faaad9a7f6d5d7af751 (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 致謝 i
中文摘要 ii Abstract iii 目錄 iv 表目錄 vi 圖目錄 vii 第一章導論 1 1. 鉀離子通道家族 1 2. 電壓調控型鉀離子通道家族 (voltage-gated potassium channel) 2 3. Kv4 鉀離子通道蛋白 (Shal-type family) 4 4. Auxiliary subunit of Kv4 channels 7 5. Kv4.3 channels相關疾病探討 9 6. 研究目的 11 第二章材料與方法 13 1. Yeast two-hybrid system 13 2. Molecular biology 19 3. Protein biochemistry 20 第三章結果 25 1. 酵母菌雙雜交系統篩選 25 2. 驗證所選出之蛋白質與Kv4.3L鉀離子通道之交互作用 29 3. 進一步探索候選蛋白在Kv4.3L之交互作用區域 33 第四章討論 35 1. 所篩選之候選蛋白與Kv4.3L鉀離子通道相互作用的結果探討 35 2. 探討候選蛋白質在細胞中已知參與之功能 37 3. 探討候選蛋白與Kv4.3鉀離子通道作用時所扮演的角色及可能參與的機制 43 4. 候選蛋白與脊髓小腦萎縮症19/22型 (SCA19/22)中Kv4.3鉀離子通道作用時所扮演的角色及可能參與的機制 47 5. Kv4.3S與Kv4.3L的比較 48 6. 未來待解決的問題及目標 49 第五章圖表說明 51 第六章參考文獻 81 表目錄 表一、此次實驗置備Kv4.3L-C terminus/ N terminus釣餌 (bait)所使用到的primer 51 表二、不同菌株與報導質體及bait搭配下之Autoactivation測試結果 52 表三、酵母菌轉殖的效率 (Efficiency test) 54 表四、五次酵母菌雙雜交實驗結果 55 表五、以Yeast Two-Hybrid所篩選到的候選蛋白質及篩選分析 56 表五補充、候選蛋白出現累積次數與對應編號 58 表六、候選蛋白與Kv4.3L之GST pull-down、Co-IP實驗統整表 60 圖目錄 圖一、以西方點墨法確認Kv4.3誘餌蛋白的表現 61 圖二、IPTG誘導細菌菌株表現GST fusion protein 62 圖三、確認bait的溶解產物表現量 63 圖四、確認bait的溶解產物與穀胱甘肽膠體微粒的結合量 64 圖五、利用共同免疫沈澱實驗與GST pull-down探討Kv4.3L與Fiz1在細胞中交互作用之可能性 65 圖六、利用共同免疫沈澱實驗與GST pull-down探討Kv4.3L與TRIP8b在細胞中交互作用之可能性 66 圖七、利用共同免疫沈澱實驗與GST pull-down探討Kv4.3L與Tctex1在細胞中交互作用之可能性 67 圖八、利用共同免疫沈澱實驗與GST pull-down探討Kv4.3L與HSC70在細胞中交互作用之可能性 68 圖九、利用共同免疫沈澱實驗與GST pull-down探討Kv4.3L與Hsp90α在細胞中交互作用之可能性 69 圖十、利用共同免疫沈澱實驗與GST pull-down探討Kv4.3L與Hsp90β在細胞中交互作用之可能性 70 圖十一、利用共同免疫沈澱實驗與GST pull-down探討Kv4.3L與Calmodulin在細胞中交互作用之可能性 71 圖十二、利用共同免疫沈澱實驗與GST pull-down探討Kv4.3L與CaMKIV在細胞中交互作用之可能性 72 圖十三、利用共同免疫沈澱實驗與GST pull-down探討Kv4.3L與CaMKII在細胞中交互作用之可能性 73 圖十四、利用共同免疫沈澱實驗與GST pull-down探討Kv4.3L與KchIP2在細胞中交互作用之可能性 74 圖十五、利用Kv4.3L其他片段探討是否與Hsp90α、Hsp90β產生交互作用之可能性 75 附圖一、Yeast Two-Hybrid之原理示意圖 76 附圖二、Yeast two-hybrid實驗中所用之Lac-Z plasmid、Bait vector及Prey vector 77 Yeast two-hybrid screening 實驗中所用之載體。 77 附圖三、KCND3結構圖 80 附圖四、Kv4.3S & Kv4.3L比較示意圖 80 | - |
dc.language.iso | zh_TW | - |
dc.title | 鑑識與電位控制型鉀離子通道Kv4.3有交互作用的蛋白質 | zh_TW |
dc.title | Identification and characterization of Kv4.3 interacting proteins | en |
dc.type | Thesis | - |
dc.date.schoolyear | 106-2 | - |
dc.description.degree | 碩士 | - |
dc.contributor.oralexamcommittee | 鄭瓊娟;卓貴美;胡孟君 | zh_TW |
dc.contributor.oralexamcommittee | Chung-Jiuan Jeng;GW Jow;Meng-Chun Hu | en |
dc.subject.keyword | 酵母菌雙雜交系統,鉀離子通道, | zh_TW |
dc.subject.keyword | yeast two-hybrid,potassium channel,Kv4.3, | en |
dc.relation.page | 96 | - |
dc.identifier.doi | 10.6342/NTU201802906 | - |
dc.rights.note | 未授權 | - |
dc.date.accepted | 2018-08-13 | - |
dc.contributor.author-college | 醫學院 | - |
dc.contributor.author-dept | 生理學研究所 | - |
dc.date.embargo-lift | 2023-10-11 | - |
顯示於系所單位: | 生理學科所 |
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