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研究生:游詩瑩
研究生(外文):Shih Ying Yu
論文名稱:鑑定並分析EIDT基因-一個會隨著紅血球成熟而大量表現之基因
論文名稱(外文):Identification and Characterization of EDIT, a Novel Gene Transcript Up-regulated during Erythroid Cell Differentiation
指導教授:林錫賢
指導教授(外文):H. H. Lin
學位類別:碩士
校院名稱:長庚大學
系所名稱:生物醫學研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
論文頁數:74
中文關鍵詞:紅血株系分化紅血株系造血造血生成
外文關鍵詞:Erythroid cell differentiationErythropoiesishematopoiesis
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紅血球生成是造血系統的一部分,並且也是一個很複雜的生物性過程,牽涉了許多轉錄因子、細胞激素刺激訊息傳遞以及協同分子的參予。在紅血球前驅細胞中,c-myb是相當重要的轉錄因子。我們先前的實驗過程中,在c-myb基因剔除的老鼠裡找到了一個新的基因-命名為紅血球分化誘導的轉錄本 (EDIT),它是個在紅血球細胞株分化過程中大量表現的基因。在本論文中,描述了老鼠EDIT基因的分子特徵。利用北方墨點法確認在MEL細胞分化過程中,EDIT基因表現是逐漸的上升。蛋白質序列比對顯示EDIT在哺乳動物與非哺乳動物間具有演化上高度保留性。次細胞組成分餾法和局部化實驗證明了EDIT分佈在細胞質與細胞核而非細胞膜。此外,免疫沈澱/西方墨點法顯示細胞質的EDIT是絲胺酸磷酸化蛋白,然而核內EDIT蛋白並無磷酸化現象。為了進一步研究EDIT的生理功能,我們已經生產並純化組胺酸-EDIT融合蛋白作為抗體製備的抗原。我們目前的工作提供EDIT初步特性的了解以及未來分析EDIT功能的方向。
Erythropoiesis is a part of hematopoiesis, which is a very complex biological process involving numerous transcription factors, cytokine signaling pathways and coregulators. C-myb is an important transcription factor in early erythroid precursors. Our previous experiments in c-myb knockout mice have identified a novel gene, named erythroid differentiation-induced transcript (EDIT), whose expression was up-regulated during erythroid differentiation. In this report, the molecular characterization of the mouse EDIT gene is described. Northern blot analysis confirmed that EDIT gene expression is gradually increased during MEL cell differentiation. Sequence alignment showed that EDIT is evolutionarily conserved among mammalian and non-mammalian species. Subcellular fractionation and localization experiments identified EDIT in cytoplasm and nucleus, but not on the membrane. Furthermore, IP/Western blot analysis showed that cytoplasmic EDIT is a phosphoserine protein, while nuclear EDIT is not phosphorylated. In order to further investigate the physiological function of EDIT, we have produced and purified His tag-EDIT fusion protein as antigen for antibody production. Our work provided preliminary characterization of EDIT and future directions for the functional analysis of EDIT.
指導教授推薦書
口試委員審定書
授權書 iii
誌謝 v
摘要 vi
Abstract vii
Table of Contents viii
Abbreviations xi
CHAPTER I Introduction - 1 -
1.1 Hematopoiesis and erythropoiesis - 1 -
1.2 Characterization of EDIT - 2 -
1.2.1 Genomic structure - 2 -
1.2.2 Protein anaysis and function - 3 -
1.3 Specific Aims - 4 -
CHAPTER II Material and methods - 5 -
2.1 Cell lines and cell culture - 5 -
2.2 MEL erythroid cell differentiation - 5 -
2.2.1 Giemsa stain - 6 -
2.2.2 Cell cycle analysis - 6 -
2.3 DNA cloning - 6 -
2.3.1 Advantage high-fidelity PCR - 6 -
2.3.2 TOPO cloning ligation - 7 -
2.3.3 Purification of DNA from agarose gels - 8 -
2.3.4 DNA ligation - 8 -
2.3.5 Transformation - 9 -
2.3.6 Colony PCR - 10 -
2.4 DNA expression constructs - 11 -
2.5 RNA methods - 11 -
2.5.1 Northern blot analysis - 11 -
2.5.2 Semi-quantitative RT-PCR - 12 -
2.6 Protein methods - 12 -
2.6.1 Western blot analysis - 12 -
2.6.2 Transient transfection - 13 -
2.6.3 Protein extraction - 13 -
2.6.4 Protein quantification - 13 -
2.6.5 Detection of the sub-cellular localization of EDIT using confocal immunofluorescence microscopy - 14 -
2.6.6 Imunoprecipitation - 14 -
2.7 Protein expression strategies by E. coli - 15 -
2.7.1 Construction of EDIT expression vector for bacteria system - 15 -
2.7.2 Expression of EDIT-His tag fusion protein - 16 -
2.7.3 Purification of recombinant protein with Ni2+ affinity chromatography - 16 -
CHAPTER III Results - 18 -
3.1 Effect of DMSO on MEL cell differentiation - 18 -
3.2 Transient transfection of EDIT to HEK 293T cells - 19 -
3.3 EDIT protein homology alignment - 21 -
3.4 Recombined EDIT protein expression in bacteria system - 21 -
3.4.1 His tag expression system - 21 -
3.4.2 GST tag expression system - 24 -
CHAPTER IV Discussion - 25 -
CHAPTER V References - 28 -
CHAPTER VI Tables - 30 -
Table I Primers used for semi-quantitative RT-PCR - 30 -
Table II Primers used for vector construction - 31 -
CHAPTER VII Figures - 32 -
Figure I-1 A process of human erythroid cell development and important factors. - 32 -
Figure I-2 Differential expression patterns of DD5-3 in Cmyb +/+, +/-, and -/- fetal livers at 13.5 and 14.5 dpc. - 33 -
Figure I-3 Hematopoietic cell lineage-restricted expression patterns of DD5-3. - 34 -
Figure 1 MEL707 differentiation. - 39 -
Figure 2 The subcellular expression of EDIT. - 40 -
Figure 3 To observe the localization of EDIT by confocal microscope- 41 -
Figure 4 Anti-myc immunoprecipitation (IP) of EDIT-myc. - 44 -
Figure 5 Sequence alignment of the human EDIT protein and orthologs in other nine species. - 46 -
Figure 6 Analysis of EDIT protein among different species. - 49 -
Figure 7 Recombined EDIT protein induced by bacteria expression system. - 50 -
Figure 8 Alternative methods to induce recombined EDIT protein. - 52 -
Figure 9 The purification of truncated EDIT by Ni2+ affinity chromatography. - 54 -
Figure 10 To find out the appropriate condition of pET28a-EDIT induction. - 56 -
Figure 11 To find out the appropriate condition of pET32a-EDIT induction. - 58 -
Figure 12 To find out the appropriate condition of induction of GST system induction. - 61 -
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