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標題: | 利用酵母菌雙雜交技術找尋在小鼠肝中與 LRH-1
有交互作用的蛋白質 Application of yeast two-hybrid screen to identify LRH-1-interacting proteins in mouse liver |
作者: | Min-Ju Hsu 許閔茹 |
指導教授: | 胡孟君 |
關鍵字: | LRH-1,酵母菌雙雜交篩選,共同免疫沉澱法,免疫螢光染色法,西方墨點法分析,啟動子活性分析, LRH-1,yeast two-hybrid screen,co-immunoprecipitation assay,immunofluorence assay,Western blotting,promoter activity assay, |
出版年 : | 2013 |
學位: | 碩士 |
摘要: | Liver receptor homolog-1 (LRH-1;NR5A2) 隸屬於NR5A 核受器家族,主要表現於肝臟、小腸、胰臟以及卵巢。LRH-1 對於發育、膽固醇的逆運輸、膽酸的恆定以及固醇類荷爾蒙的生成等扮演重要的角色。先前的研究指出,LRH-1 會和調控因子產生交互作用,進而調控許多下游基因的表現;然而,目前已知與 LRH-1 有交互作用的蛋白質並不多,因此我們希望藉由酵母菌雙雜交篩選,尋找與LRH-1
有交互作用的蛋白質,並且進一步探討它們調控 LRH-1 的功能。 將 LRH-1 建構至 LexA DNA binding domain (DBD) 下游,形成誘餌蛋白,針對建構於 GAL4 activation domain (AD) 的小鼠肝臟的 cDNA library 進行篩選。共得到58 種不同的蛋白質,從中挑選出7個蛋白質,分別為H3 histone, family 3B (H3f3b)、Methyl-CpG binding domain protein 1 (Mbd1)、Zinc finger protein 579 (Zfp579)、MYC-associated zinc finger protein (Maz)、Zinc finger and BTB domain containing 44 (Zbtb44)、Ring finger protein 128 (Rnf128) 和 Nuclear receptor subfamily 1 group H, member 4 (Nr1h4, FXR)。經由共同免疫沉澱法,證實這七個蛋 白質皆與 LRH-1 有交互作用。接著利用免疫螢光染色法,發現多數的蛋白質主要分佈於細胞核,與 LRH-1 的螢光表現重疊。後續經由功能性分析 (functional assay),包括: 西方墨點法分析以及啟動子活性分析,觀察蛋白質是否會影響 LRH-1 蛋白質的表現量或是影響 LRH-1 調控啟動子的轉錄活性。 Liver receptor homolog-1 (LRH-1; NR5A2) is a member of the nuclear receptor NR5A subfamily. LRH-1 is predominantly found in liver, intestine, pancreas and ovary.In these tissues, LRH-1 plays a critical role in development, reverse cholesterol transport, bile-acid homeostasis and steroidogenesis. Previous studies have shown that LRH-1 can interact with coregulators and regulate the expression of many downstream genes. However, there are only a few proteins which were known as LRH-1-interacting proteins. Therefore, we carried out a yeast two-hybrid screen to identify proteins that will interact with LRH-1 and investigate their physiological functions with LRH-1. LRH-1 was fused to the LexA DNA binding domain (DBD) as a bait, and performed screening of the mouse liver cDNA library fused to a GAL4 activation domain (AD). Eventually, we identified 58 proteins and chose 7 of them for further investigation. These proteins are H3 histone, family 3B (H3f3b), Methyl-CpG binding domain protein 1 (Mbd1), Zinc finger protein 579 (Zfp579), MYC-associated zinc finger protein (Maz), Zinc finger and BTB domain containing 44 (Zbtb44), Ring finger protein 128 (Rnf128) and Nuclear receptor subfamily 1 group H, member 4 (Nr1h4, FXR). The co-immunoprecipitation assay verified that all of the proteins interact with LRH-1. Moreover, the immunofluorence assay results demonstrate that most of the proteins are colocalized with LRH-1 in the nucleus. We further performed the functional assay, such as: Western blotting and promoter activity assay, to identify whether these proteins will affect LRH-1 protein expression or modulate the promoter activity through LRH-1. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/61135 |
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顯示於系所單位: | 生理學科所 |
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