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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24340完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 胡孟君 | |
| dc.contributor.author | Chien-Ting Pan | en |
| dc.contributor.author | 潘建廷 | zh_TW |
| dc.date.accessioned | 2021-06-08T05:22:30Z | - |
| dc.date.copyright | 2005-08-02 | |
| dc.date.issued | 2005 | |
| dc.date.submitted | 2005-07-25 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/24340 | - |
| dc.description.abstract | CYP11A1基因的產物為膽固醇側鍵截切酶(cholesterol side-chain cleavage enzyme, P450scc)。 P450scc酵素在類固醇荷爾蒙的合成途徑中，負責催化第一個步驟，也就是將膽固醇轉換成孕烯醇酮 (pregenelone)。CYP11A1普遍存在於腎上腺、性腺等所有類固醇生成組織中。Liver receptor homolog-1 (LRH-1)為 NR5A家族中的一員，為一轉錄因子，其大量存在於某些類固醇生成組織內，例如卵巢，並具有調控類固醇生成基因的能力。
我們首先使用大腸桿菌表現系統大量產生小鼠LRH-1 (mLRH-1)兩個不同片段的胜肽，分別是具有N端1~115胺基酸序列及C端169~560胺基酸序列。經電泳純化後，以此製備LRH-1抗體。在西方墨染法中，以所製備的LRH-1抗體，證實LRH-1存在於黃體的初代培養細胞中。293T細胞株經轉染mLRH-1表現質體後，亦能以此抗體偵測到mLRH-1的表現。我們利用共同轉染的方法研究LRH-1調控CYP11A1基因表現的能力。LRH-1對於1.5 kb長度的CYP11A1啟動子，即具有增進其轉錄的作用。CYP11A1基因上的兩個SF-1結合序列(-40和-1600)，經由點突變後發現，位於-40上的SF-1結合序列，可能是LRH-1在CYP11A1基因啟動子上的主要結合序列。轉染Dax-1後，LRH-1增進CYP11A1基因啟動子的作用，會受到抑制。LRH-1會和轉譯後修飾蛋白SUMO-1產生鍵結，而lysine 289為其主要的結合位置。SUMO-1和LRH-1結合後，會抑制LRH-1的轉錄活性，其抑制效果和Dax-1並無關聯。我們發現LRH-1在轉染細胞中的表現並不穩定，當加入proteasome抑制劑MG-132後，可提升其蛋白含量。 | zh_TW |
| dc.description.abstract | CYP11A1 gene encodes P450scc enzyme, which controls the first and rate-limiting step of steroidogenesis by conversion of cholesterol to pregnenolone. CYP11A1 gene is expressed in many steroidogenic tissues like adrenal and gonad. Liver receptor homolog-1 (LRH-1) is a transcription factor and belongs to a member of nuclear receptor NR5A family. LRH-1 is expressed in some steroidogenic tissues such as ovary and may play a role in the regulation of steroidogenic gene.
To study the function of LRH-1, two peptides of mouse LRH-1 were produced in E. coli for the generation of antibody. This antibody was used to detect the presence of LRH-1 in rat primary luteal cells by Western blotting. Transfection of LRH-1 expression vector in 293T cell line also can be detected by Western blotting. To examine the ability of LRH-1 in the regulation of CYP11A1 gene expression, we cotransfected LRH-1 expression vector and human CYP11A1 promoter construct into 293T cell line. LRH-1 can enhance the shortest 1.5 kb CYP11A1 promoter activity. Two functional SF-1 binding sites were located at -40 and -1600 in CYP11A1 promoter. By mutating the SF-1 binding sites, we found that SF-1 binding site located at -40 is the major site of LRH-1 binding to CYP11A1 promoter. Induction of CYP11A1 promoter activity by LRH-1 was inhibited by Dax-1. LRH-1 can be conjugated with post-translation modification protein SUMO-1 and we found that lysine 289 is the major conjugation site of SUMO-1. When LRH-1 was conjugated with SUMO-1 the ability of LRH-1 inducing CYP11A1 promoter activity was inhibited and that was independent of Dax-1 function. We found that LRH-1 may not be stable in the transfected cell.The proteasome inhibitor, MG-132, can increase the amount of LRH-1 protein in transfected cell. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T05:22:30Z (GMT). No. of bitstreams: 1 ntu-94-R92441010-1.pdf: 1006745 bytes, checksum: a02441f2636b56d208f55ec44ef14a95 (MD5) Previous issue date: 2005 | en |
| dc.description.tableofcontents | 目錄.....................................................Ⅰ
圖表目錄.....................................................Ⅲ 中文摘要.....................................................Ⅳ 英文摘要.....................................................Ⅴ 第一章、序論..................................................1 一、類固醇荷爾蒙的生成.......................................1 二、CYP11A1基因............................................2 三、CYP11A1啟動子特性......................................2 四、轉錄因子SF-1............................................4 1、 SF-1之特性............................................4 2、 SF-1的表現............................................4 五、轉錄因子LRH-1.........................................5 1、 LRH-1蛋白特性........................................5 2、 LRH-1於胚胎發育及分化上的角色........................6 3、 LRH-1的作用..........................................6 六、影響LRH-1作用之因子....................................8 1、 輔因子................................................8 2、 轉譯後修飾蛋白SUMO-1的作用.........................9 七、研究動機................................................10 第二章、材料與方法............................................11 一、質體構築................................................11 二、重組蛋白於大腸桿菌內表現的誘導及分離....................15 三、重組蛋白質純化..........................................16 四、抗體....................................................16 五、SDS-PAGE..............................................17 六、細胞培養................................................19 七、暫時性轉染法............................................19 八、Luciferase活性分析.......................................20 九、胞外轉錄和轉譯反應及胞外sumoylation作用.................20 第三章、結果..................................................22 一、mLRH-1抗體的製備......................................22 1、 mLRH-1蛋白的構建與表現..............................22 2、 重組mLRH-1蛋白之純化................................23 3、 mLRH-1抗體測試......................................23 二、 LRH-1調控人類CYP11A1基因之功能.......................24 1、 LRH-1對於CYP11A1啟動子之影響.......................24 2、 LRH-1在CYP11A1啟動子上的結合序列...................25 三、影響LRH-1調控人類CYP11A1基因轉錄活性之因素..........25 1、 LRH-1上N端1-101胺基酸對於CYP11A1啟動子之作用......25 2、 Dax-1抑制LRH-1增進CYP11A1啟動子之作用..............26 四、轉譯後修飾蛋白SUMO-1對於LRH-1之影響.................26 1、 LRH-1上的SUMO-1結合位置............................27 2、 SUMO-1的轉譯後修飾作用對LRH-1調控CYP11A1之影響...28 3、 SUMO-1和LRH-1結合後之抑制效果與Dax-1無關..........28 五、LRH-1蛋白表現之穩定性.................................29 第四章、討論..................................................30 參考文獻.....................................................35 圖...........................................................43 圖表目錄 圖一、 人類CYP11A1啟動子上順式調節元素之位置...............43 圖二、 mLRH-1蛋白在M15菌株的表現........................44 圖三、 mLRH-1蛋白於M15菌株表現之確認.....................45 圖四、 mLRH-1-C及mLRH-1-N蛋白之純化.....................46 圖五、 anti-mLRH-1-N及anti-mLRH-1-C抗體於大腸桿菌 表現mLRH-1蛋白之測試................................47 圖六、 anti-mLRH-1-N及anti-mLRH-1-C抗體於細胞表現 mLRH-1蛋白之測試....................................48 圖七、 LRH-1增進人類CYP11A1啟動子轉錄活性.................49 圖八、 LRH-1與SF-1增進不同片段長度之人類CYP11A1 啟動子轉錄活性.......................................50 圖九、 SF-1結合序列對LRH-1與SF-1調控人類CYP11A1 啟動子的影響.........................................51 圖十、 N端刪除A/B domain不影響mLRH-1調控人類 CYP11A1啟動子能力...................................52 圖十一、Dax-1抑制LRH-1促進人類CYP11A1啟動子 轉錄活性之作用.......................................53 圖十二、mLRH-1會和SUMO-1結合.............................54 圖十三、mLRH-1與SUMO-1結合之lysine位置....................55 圖十四、SUMO-1的結合對mLRH-1調控CYP11A1活性之影響.......56 圖十五、SUMO-1的結合對mLRH-1調控能力之影響...............57 圖十六、SUMO-1抑制LRH-1活性與Dax-1無關...................58 圖十七、MG-132增加mLRH-1蛋白表現量........................59 | |
| dc.language.iso | zh-TW | |
| dc.subject | 轉錄因子 | zh_TW |
| dc.subject | 轉譯後修飾蛋白 | zh_TW |
| dc.subject | LRH-1 | en |
| dc.subject | SUMO-1 | en |
| dc.subject | CYP11A1 | en |
| dc.title | LRH-1抗體製備及LRH-1調控CYP11A1之研究 | zh_TW |
| dc.title | Preparation of LRH-1 Antibody & Regulation of CYP11A1 Gene by LRH-1 | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 93-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 鍾邦柱,張繼堯,郭應誠 | |
| dc.subject.keyword | 轉錄因子,轉譯後修飾蛋白, | zh_TW |
| dc.subject.keyword | LRH-1,CYP11A1,SUMO-1, | en |
| dc.relation.page | 59 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2005-07-26 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 生理學研究所 | zh_TW |
| 顯示於系所單位： | 生理學科所 | |
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