TSG101是史丹福大學Stanley Cohen實驗室所發現之腫瘤易感基因，在各種人類的癌組織細胞中，雖無TSG101 genomic DNA上的異常，但會有許多異常的轉錄產物，並有報告指出腫瘤易感基因TSG101會參與MDM2/p53的回饋控制，細胞膜運輸、接受器回收的調控，亦參與細胞內轉錄活性之調控，然而詳細機制還不是很清楚，為探討TSG101與上述功能之相關性，並定位TSG101蛋白subcellular localization，所以我們將TSG101的cDNA區分成數個不同的片段，並與pEGFP載體形成重組質體，送入細胞中表現，以西方墨點分析確定可表現預期大小的蛋白後，再以螢光顯微鏡及共軛焦顯微鏡進行觀察，結果顯示TSG101全長蛋白分佈在內質網、高基氏體及endosome等胞器內，而其氨基酸136-233及316-390區域對於TSG101在細胞的定位上扮演很重要的角色。此外，有報告指出TSG101與OP18有交互作用，TSG101在細胞週期的M時期會在紡錘絲上出現，OP18又與紡錘絲的形成有關，因此為探討TSG101與OP18之間在M時期的分佈情形，首先，將OP18基因加以選殖，並表現GST-OP18融合蛋白，用以製備抗血清，並將OP18 cDNA與載體pDsRed1-C1形成重組質體後，經以OP18抗體進行西方墨點分析，確定重組質體可在轉染的細胞內表現pDsRed-OP18融合蛋白，在將此OP18/pDsRed重組質體與TSG101(1-390)/pEGFP表現質體進行共同轉染，並以nocodazole使細胞停在M時期進行觀察，結果顯示，OP18宏色螢光均勻散佈在整個細胞內，但TSG101綠色螢光則呈點狀分佈在細胞質及染色體上，至於，TSG101及OP18是否有交互作用，有待進一步研究加以釐清。

TSG101 was identified as a tumor susceptibility gene by Stanley Cohen. In a variety of human cancers, no genomic deletion in TSG101 gene has been reported but many aberrant TSG101 transcripts has been found. Some studies have revealed that TSG101 participates in MDM2/p53 regulatory circuitry、membrance trafficking and receptor recycling. Other reports also showed that TSG101 might be a transcription regulatory factor. However, mechanism of these TSG101 function awaits further characterization.
To further scrutinize the function of TSG101 and its subcellular localization, a varieties of GFP-based recombinant plasmids which contain various length of TSG101 cDNA have been constructed and transfected into cells. Western blot analysis had shown that these constructs could express GFP-TSG101 fusion protein of expected size. The fluorescence and confocal microscopy have shown that wild type TSG101 localized in ER, Golgi and endosome compartments, also amino acid residues 136-233 and 316-390 of TSG101 are two important regions for its subcellular localization.
Previous reports had shown that TSG101 interact with OP18 which is an important regulator for spindle formation in M phase. To elucidate the localization of TSG101 and OP18 in M phase cell, we have cloned OP18 and generate GST-OP18 fusion protein for anti-OP18 antiserum production.Then, pDsRed-OP18 fusion protein expressed in OP18/pDsRed recombinant plasmid transfected cell was detected by western blotting analysis using this anti-OP18 antiserum. The subcellular localization of DsRed-OP18 and GFP-TSG(1-390) fluorescence were recorded in double transfected cells which were arrested in M phase by nocodzole treatment. We observed the evenly distribution of pDsRed-OP18 red fluorescence and punctate vesicular localization of GFP-TSG(1-390) green fluorescence. Whether these two protein interact functionally awaits further investigation.

中文摘要 2
英文摘要 4
前言 6
材料與方法 11
結果 29
討論 36
參考文獻 44
圖表 49

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