TSG101(Tumor susceptibility gene)為一多功能的腫瘤易感基因，其基因產物為含有390個氨基酸，分子量約46 kDa的蛋白質，主要存在於細胞質。關於TSG101在細胞內功能之研究已很多，文獻指出TSG101具有調控細胞週期、參與膜蛋白運輸、調節轉錄活性等功能，為瞭解TSG101是透過哪些胞內蛋白而達成調控上述功能的目的，本論文以酵母菌雙雜交系統篩選與TSG101交互作用的蛋白。首先將建構好之TSG101/pAS2-1重組質體轉形至YRG2酵母菌株用以當釣餌，進行酵母菌雙雜交系統之cDNA基因庫篩選。經篩選後獲得68種有交互作用之蛋白，經NCBI序列比對辨認出17種已知的蛋白，將之以功能分類可初略分為與細胞週期調控、蛋白質運輸、調節轉錄活性、蛋白質修飾、訊息傳遞及其他生理功能相關等蛋白。以上結果除再次呼應前人研究成果之外，並提供若干與TSG101功能相關的題材供後續TSG101相關生物功能之探討。此外，為探討TSG101在核內功能為何，我們選取與調節基因表現有關的KLIP1為材料，以pull-down assay及免疫共沈澱法進一步證明兩者在哺乳動物細胞內確有交互作用存在，未來將可進一步研究KLIP1功能及其與TSG101間相互作用之相關生物功能機轉。

Tumor Susceptibility Gene, TSG101, has been identified as a tumor susceptibility gene with multiple functions. TSG101 encodes a 46 kDa protein composed of 390 amino acids. As previous studies reported, TSG101 participates in cell-cycle control, membrane proteins’ trafficking, and transcriptional regulation. To identify the proteins that mediated function involved TSG101, we perform yeast two-hybrid cDNA library screening to search for TSG101-interacting proteins. A construct pAS2-1-TSG101 was used as a bait to screen a human testis cDNA library. This screening selected 68 TSG101 interacting clones, including 17 known proteins. These proteins were functionally classified as participating in cell-cycle alteration, protein sorting, transcriptional regulation, modification, signal transduction and other functions. Our results provide the evidences which not only confirm the results of previous studies, but also provide further information related to TSG101 biological functions worth intensive study. Among these clones, we choose KLIP1 gene, which encodes a transcription factor, for further study to elucidate the functional role of TSG101 in nucleus. In vitro GST pull-down assay and in vivo co-immunoprecipitation assay were performed using GST-KLIP1 and HA-tagged KLIP1, respectively, have demonstrated that TSG101 and KLIP1 indeed interact with each other within mammalian cells. Detailed biological function mediated through this TSG101 and KLIP1 interaction awaits further investigation.

中文摘要 1
英文摘要 2
前言 3
材料與方法 8
結果 29
討論 42
參考文獻 48
圖表 55

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