本論文主要探討由本研究室所建立之吳郭魚(Oreochromis mossambicus)腦發育初期的表現序列標籤資料庫(expressed sequence tags, ESTs)其中一條與細胞增生相關的基因，Transducer of ErbB2,1(Tob1)。Tob1是Btg/Tob protein family內的一種蛋白質，具有抑制細胞增生的一種功能性蛋白質。本論文針對Tob1進行生物資訊分析。開放閱讀區(ORF)之分析結果，為片段長度1020 bp，可轉譯出339個胺基酸序列。另，利用NEBcutter V2.0 對tob1開放閱讀區進行限制酵素的切位分析，並以真核系統表現具螢光的Tob1蛋白質，將Tob1基因建構在綠色螢光蛋白載體中(green fluorescent protein, GFP)，並轉染至老鼠瘤神經細胞(Neuron-2a, N2a)中，利用共軛焦顯微鏡觀察細胞內Tob1蛋白質分布情形，結果顯示pEGFPN1-Tob1在N2a細胞質中有GFP綠色螢光表現，且細胞質有綠色螢光顆粒狀聚集的現象。進一步，建構原核生物表現系統，將建構好之pRSETa-Tob1載體利用BL21 E.coli來表現其蛋白質，並藉由標定6X-His Tag以親和性管柱層析(affinity chromatography)的方法，進行蛋白質之純化，純化後的Tob1蛋白質投予在N2a細胞，進行增生影響測試，利用MTT做分析，結果發現投予Tob1蛋白質會影響細胞的增生。

The expressed sequence tags, (ESTs) derived from the developing tilapia brain, were established in our lab. There are 1092 transcripts. A transcript out of 1092 transcripts of ESTs was identified as a partial fragment of Transducer of ErbB2,1(Tob1). The unique gene, Tob1 encode a member of Btg/Tob protein family and play a role in cell growth. Bioinformatics were searched for Genbank: EE724041.1(Tob1), which is a gene with 1020 bp of open reading frame(ORF), and deduced as 339 amino acid of protein sequence. The pEGFPN1-Tob1 plasmid constructed by inserting tob1 into pEGFPN1 vector, was transfected into mouse neuroblastoma cell (neuron-2a cell, N2a). The distribution of Tob1 expressed in pEGFPN1-Tob1 transfected N2a cell was observed by confocal microscopy. This result shows that Tob1 protein is distributed in the cytoplasm of N2a. Tob1 protein was expressed by BL21(Escherichia coli), a prokaryotic system, and purified with Ni-NTA affinity chromatography. The effects of Tob1 protein on cell proliferation were investigated. These results show that the proliferation of neuro-2a cells is affected by Tob1.

論文審定書………………………………………..………………………..i
誌謝………………………………………………………………………...ii
中文摘要…………………………………………..………………………iii
英文摘要………………………………………………….……………….iv
目錄………………………………………………………………………...v
圖次………………………………………………………………………..vi
前言……………………………………………………….………………..1
實驗材料與方法…………………………………………...………………3
結果……………………………………………………….………………23
討論………………………………………………………….……………27
參考文獻……………………………………………………….…………29
附圖(表)………………………………………………………….……….33

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