臺灣博碩士論文加值系統

本實驗室之前研究指出，溫度影響吳郭魚之性別分化及腦神經的
發育，而本實驗室發展孵化後10天吳郭魚腦基因的表現，並建立吳郭魚腦表現序列標記( expressed sequence tag , EST)資料庫。而編號F10A83為一個低溫誘發表現之基因，F10A83的cDNA全長為1526 bp，轉譯之蛋白質由176個氨基酸所組成，為一個富含甘氨酸的蛋白質( glycine-rich protein )。本研究藉由6 個His作為標記，將F10A83開放閱讀區( open reading frame, ORF )建構於原核表現系統中，再利用親和性管柱將F10A83成功分離純化，所分離之F10A83蛋白質針對吳郭魚腦細胞，探討其生長趨勢分析。在真核生物方面，將F10A83之ORF構築在綠螢光蛋白( green fluorescent protein, GFP )表現載體上，轉染( transfection )至老鼠神經瘤細胞( Neuro-2a )中，使用螢光顯微鏡觀察發現，F10A83大部分都表現於細胞核中，為細胞核蛋白的一種。我們也篩選穩定表現F10A83之Neuro-2a細胞，探討分析F10A83基因對細胞生長的影響。

In tilapia , sex determination is controled by genetic and triggered by the environmental factors. Expressed sequence tags ( EST ) derived from the developing tilapia brain is cloned in our lab. The cDNA full length of the gene, F10A83 was cloned. In the present study, F10A83 is a gene with 1526 bp of cDNA sequence, and deduced 176 amino acids of protein sequence. F10A83 was overexpressed as a GFP fusion protein in mouse neuroblastoma Neuro-2a cells. F10A83 is abundantly distributed in the nucleus of Neuro-2a cell. The protein of F10A83 was expressed in the prokaryotic cell (BL21) and eukaryotic cell (neuro-2a), and purified using a simple purification process, inducing, isolation, and Ni-NTA affinity chromatography. The protein of F10A83 in both E. coli system and neuro-2a cell line expression has been established.

頁次
致謝---------------------------------------------------- 1
中文摘要-------------------------------------------------2
英文摘要-------------------------------------------------3
目錄-----------------------------------------------------4
附圖、表目錄---------------------------------------------5
前言-----------------------------------------------------6
實驗材料與方法-------------------------------------------9
結果----------------------------------------------------41
討論----------------------------------------------------45
參考文獻------------------------------------------------49
附圖、表------------------------------------------------53

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