臺灣博碩士論文加值系統

本論文的研究目標為建立調控組的整體快速鑑別技術平台，承續前人研究以人類肝臟腫瘤細胞株所標識之探針結合篩選人類肝臟互補核酸表現高度專一性酵母菌株以得到對側因子群(Trans factors)，再利用現有的對側因子篩選對於抗腫瘤基因p53調控相關的同側序列群(Cis element)以建構整個調控組搜尋技術完整性。本項研究以酵母菌p53轉形株純化型蛋白質轉錄因子循環式擴增選拔目標(CASTing, Cyclic Amplification and Selection of Targets)核酸序列，經單雜合篩選系統建立結合核酸共識序列，證實整體調控組減除型鑑別分析技術平台的可行性以及提高相對速效性與專一性。
在蛋白質純化系統建立上，利用陰離子交換之HQ column:strangly basic anion exchanger,-N+(CH3)3使蛋白質結合再利用不同鹽濃度之溶劑進行洗取，而得到目標蛋白，即由酵母菌株表現純化型轉錄因子蛋白質p53，在由此蛋白質進行CASTing核酸製備，篩選轉錄因子與CASTing核酸結合複合體，獲得轉錄因子專一性同側序列群CASTing核酸模板，選殖序列群構築於單雜合酵母菌系統ß-gal報導基因載體pLacZi，轉形表現轉錄因子酵母菌株勝任細胞，塗盤進行酵母菌株X-gal藍白篩選，轉形成功之菌株其嵌入之核酸序列會與p53轉錄因子進行專一性結合而使基因表現產生b-galactosidase分解X-gal而呈藍色反應，定序藍色酵母菌株報導載體選殖構築的CASTing核酸片段序列，比對分析獲得共識結合序列。
本項論文研究初步證實整體調控組減除型鑑別分析技術平台的可行性以及相對提高快速性與專一性，最大特色則是具備同時鑑別分析多重未知調控序列目標與對應調控因子的效能。

The goal of this thesis is to establish fast and global analysis platform of differential regulomics , it continue futher study with probe of labeling human tumor cell line to screening human liver complementary nucleic acid from high expressing and specific yeast yield to obtain Trans -factors, and then utilize existing Trans -factors selection regulation relevantly to the Cis element of antitumor gene p53 by building and constructing the screening regulomics technological integrality entirely. This research is purification transcription factor p53 protein from transform yeast cell line with Cyclic Amplification and Selection of Targets to obtain nucleic acid, finding consensus sequence of transcription factor binding from one hybride selection system,verify that adjusts regulomics subduction type and distinguishes the feasibility which analyses the technological platform and improves the relatively quick-acting and specific.
In the building of protein purification system , utilize HQ column of strangly basic anion exchanger, -N+(CH3)3 to bind protein and then utilize different concentration of salt solvent to elute, and receive the goal protein, namely is purification transcription factor protein p53 by the expression yeast,by this protein CASTing the nucleic acids, selection complex of CASTing nucleic acid and transcription factor, obtain specific transcription factor of CASTing nucleic acid template , insert nucleic acid template to construct on b-gal reporter gene pLacZi of yeast , transform to the expressing transcription factor yeast competent cell , plating and X-gal blue- white selection, the transformation success yeast could make gene expression yield β-galactosidase to destroy X-gal and blue response, sequenceing CASTing nucleic acid from blue response yeast of clone construct reporter plasmid , compare and analysing to obtaining the consensus sequence.
The thesis study has accomplished the preliminart feasibility of the differential subtractive analysis of global regulomics with performance features on simultaneous identification of multiple unknown targets at higher speed .

目 錄
頁次
誌謝 …………………………………………………………. I
中文摘要 …………………………………………………………. II
英文摘要 ………………………………….……………………… III
目錄 …………………………………………………………… V
圖目錄 …………………………………………………………… IX
表目錄 …………………………………………………………… XI

第一章 緒論 ………………..…………………………………….. 1
第一節 研究背景 …………………………………………….. 1
第二節 研究動機 ……………………………………………... 2
第三節 章節摘要 ……………………………………………... 3

第二章 轉錄因子蛋白質之純化 ………………….……………. 4
第一節 蛋白質純化及分離方法 ……...………………… 4
第二節 搜尋轉錄因子互補核酸片段 ………...……………… 5
一、酵母菌株蛋白質表現系統 ………..……………………. 5
二、酵母菌的背景歷史與研究應用 ……………..……….. 5
三、酵母菌系統菌株MaV203 Yeast …...…………………. 7
第三節 實驗材料 …………………………………………… 7
一、人類互補核酸大腸桿菌表現集存庫ProQuest cDNA Library …...…… 7
二、酵母菌野生型菌株Pichia pastoris Yeast ……...… 10
三、配製聚丙烯醯胺SDS-PAGE凝膠電泳溶液 …...…… 12
第四節 實驗方法與結果 ……………………………….….… 16
(一)未知菌株：搜尋轉錄因子互補核酸片段 …….….. 16
一、探針篩選及呈色 …….….. 16
1. 標識DIG探針的檢測方法DIG Detection …….….. 16
2. 轉漬膜的速凍與解凍處理Blot Freeze & Thaw …….….. 16
3. 探針結合篩選Probe binding screening ……….. 17
4. 探針DIG呈色反應DIG Wash and Block Buffer ……….. 18
二、酵母菌落質體製備與擴增Plasmid Prep ………... 19
1. 酵母菌落質體製備 ……… 19
2. 酵母菌落質擴增 ……… 21
三、酵母菌落擴增核酸片段定序確認Sequencing ……… 22
(二)已知菌株：純化對側因子p53 …………….………….. 22
一、蛋白質粗萃取 ………………….……………. 22
二、SDS丙烯醯胺凝膠電泳 …………………………… 23
三、管柱層析純化目標蛋白質 …………………………… 25
第五節 蛋白質驗證 …………………………… 31
(一)Dot immunostain …………………………….………...... 31
(二)探針結合呈色 …………………………….………….. 32
一、p53核酸探針3’端DIG (Digoxigenin)標識 …………… 32
1. 探針DIG標識系統 …………… 32
2. 探針3’端DIG標識技術方法 …………………………… 32
(1) 核酸標識DNA labeling ……………………………..... 32
(2) 寡核苷酸標識Oilgonucleotide labeling ………….. 33
二、核酸擴增模版的製備 ………….. 33
1. 核酸擴增模版的前期聚合酶鏈反應Pre-PCR ………….. 33
2. 前期聚合酶鏈反應產物之電泳分析 ………….. 34
3. 前期聚合酶鏈反應產物之電泳洗出Electroeluction ………….. 35
4. 前期聚合酶鏈反應產物之OD值 ………….. 35
5. 探針3’端DIG標識DIG-3´-label ………………… 36
6. 探針結合篩選Probe binding screening ………………… 37
7. 探針DIG呈色反應DIG Wash and Block Buffer ….…….. 38
第六節 討論 …………………………………….…………….. 40

第三章 序列目標循環式擴增選拔CASTing ………………. 42
第一節 序列目標循環式擴增選拔CASTing的技術及應用 ………………. 44
第二節 實驗材料 ……………………………………… 46
一、對側轉錄因子製備 ……………………………………… 46
二、擴增引子設計 ……….…………………. 47
三、核酸擴增模版的製備 ……….…………………. 47
1. 核酸擴增模版的前期聚合酶鏈反應Pre-PCR …….……………………. 47
2. 前期聚合酶鏈反應產物之電泳分析 ……… 48
3.前期聚合酶鏈反應產物之電泳洗出Electroeluction ……… 49
4. 前期聚合酶鏈反應產物之OD值 ………………………... 49
第三節 實驗方法與結果 ………………………... 50
1. 序列目標循環式擴增選拔第1回合CASTing-1 …………….………….. 50
2. 序列目標循環式擴增選拔競爭減除型第2~5回合 ………….…………….. 51
第四節 討論 ………….…………….. 52

第四章 報導基因之基因表達 ………………….……………. 54
第一節 報導基因的分析方法種類 …………….……………. 54
第二節 酵母菌雜合系統 …………………….…………….. 56
第三節 實驗材料 ……………………………………… 56
第四節 實驗方法與結果 ……………………………………… 58
一、酵母菌勝任細胞菌株YM4271 Yeast Compotent Cell製備 ……………….………… 58
二、pLexA-53質體製備 ……….………….……… 59
三、重組質體 …………………………….……………….. 60
1.載體處理 ………………………………………………… 60
2. insert DNA處理進行ligation …………………………… 61
四、載體轉形 ………………………………………………. 62
第五節 討論 ………………………………………………….. 63

第五章 結論與展望未來 ……………………………………… 64

參考文獻 …………………………………………………….. 76











圖 目 錄
頁次
圖2-1、 Comments for pPC86(no insert) 7093 nucleotide …………………… 8
圖2-2、 pPC86 vector完整序列 …………………………………………….…. 9
圖2-3、 NBT/BCIP呈色結果 ……………………………………………….. 18
圖2-4、呈色酵母菌落選取確認Colony Selection …………………… 19
圖2-5、酵母菌落質體製備進行1 % 瓊脂膠體電泳確認 …………………… 20
圖2-6、瓊脂膠體電泳確認酵母菌落擴增核酸片段 ………………...……..… 21
圖2-7、 BSA Curve ……………….………………………… 23
圖2-8、粗萃取物SDS-PAGE電泳圖 …………………………………….…… 24
圖2-9、高效能蛋白質純化系統BioCAD 700E ………………….…………… 25
圖2-10、層析流程圖 …………………………………….…………… 25
圖2-11、蛋白質吸光圖譜比較 ………………………………….……………… 28
圖2-12、蛋白質圖譜 ……………………………………………….………… 29
圖2-13、實驗組與空白對照組圖譜 …………………………………………… 30
圖2-14、immunostain …………………………………………………………. 31
圖2-15、p53 prePCR電泳圖 …………………………………………………… 34
圖2-16、探針標誌效率結果 …………………………………………………… 37
圖2-17、 NBT/BCIP呈色結果 ………………………………………….……… 39
圖3-1、 p53蛋白質結構圖 …………………………………………………… 47
圖3-2、前期聚合酶鏈反應產物之電泳分析 …………………………………… 48
圖4-1、酵母菌單雜合系統 ………………...…………………………………… 56
圖 4-2、pLexA-53 載體特徵輿圖 ……………………………………………... 57
圖4-3、 pLacZi載體特徵輿圖 ……..………………………………………… 57
圖4-4、表現轉錄因子酵母菌株之勝任細胞 ……………………………….… 59

圖4-5、以限制酶EcoR I 和Sal I切割過的pLacZi膠體電泳確認.....................60


















表 目 錄
頁次
表2-1、緩衝液配製成分 …………………………….…………...…………… 15
表2-2、結合(binding)溶液成份 …………………………….……..…………… 17
表2-3、BSA standard OD值 ………………………….……..……………… 22
表2-4、蛋白質萃取物濃度 ……………………………….……….………… 23
表2-5、純化菌種及層析方式參數表 ……………………….………….……… 26
表2-6、蛋白質收集液片段比較表 …………………………….……………… 29
表2-7、各蛋白質洗取時間比較表 …………………………………….……………… 30
表2-8、Pre-PCR DNA O.D值 ………………..…………..…………………… 35
表2-9、DIG Dilution series table ……………………………………………… 36
表2-10、結合(binding)溶液成份 ……………………………………………… 38
表3-1、Pre-PCR DNA O.D值 ………………………………………………… 49
表3-2、10x CBS CASTing Buffer製備 ………..……………………………… 51
表4-1、處理過後載體O.D值 ……………………………………….. 61
表4-2、insert DNA 經過klenow polymerase處理後O.D值 ……………………………………….. 61
表4-3、insert DNA 經過T4 polynucleotide kinase處理後O.D值 ……………………………………….. 62

陳思妏. (2004): 真核細胞基因表現之整體調控組差異鑑別. 中國文
化大學生物科技研究所碩士論文.

方維霖. (2002): 建構高效率蛋白質純化製程. 逢甲大學化學工程研究所碩士論文.

王立禾, 阮立昂 ( 1977)： 蛋白質構造與功能. 復漢出版社, 台南市.

Andrew J.W., James S.D., Evelyn S., Daniela U., and Lawerence J. A. (2002)： A Proteomics Approach for the Identification of DNA Binding Activities Observed in the Electrophoretic Mobility Shift Assay. Molecular & Cellular Proteomics 1.6:472-478

Bollag D.M., Rozycki M.D., and Edelstein S.J. (1996)： Protein Methods, Second Edition ed. Wiley-Liss, New York, NY.

Byrne M. P., Smith T. J., Montgomery V.A., and Smith L.A. (1998)： Purification, Potency, and Efficacy of the Botulinum Neurotoxin Type A Binding Domain from Pichia pastoris as a Recombinant Vaccine Candidate. Infection and Immunity, 4817–4822

Causier B., Davies B. (2002)： Analysing protein-protein interactions with the yeast two-hybrid system. Plant Molecular Biology 50, 855–870

Chen P.L.,Chen Y. M., Bookstein R.,and Lee W. H. (1990)：
Genetic mechanisms of tumor suppression by the human p53 gene.Science 250:1576-1580
Chang C.F., Gallia G.L., Muralidharan V., Chen N.N., Zoltick P., Johnson E., and Khalili K..Evidence that Replication of Human Neurotropic JC Virus DNA in Glial Cells Is Regulated by the Sequence-Specific Single-Stranded DNA-Binding Protein Purα. Journal of Virology 70,4150-4156

David Nelson (2001)： Yeast Genetic Screens, Two Hybrid, One Hybrid,Synthetic Lethal and YACs. Lecture 2 on Yeast Genetics

Diatchenko L., Lau Y.C., Campbell A.P., Chenchik A., Moqadam F., Huang B., Lukyanov S., Lukyanov K., Gurskaya N., Sverdlov E.D., and Siebert P.D. (1996)： Suppression subtractive hybridization:A method for generating differentially regulated or tissue-specific cDNA probes . Biochemistry , Vol.93:6025-6030

Denobel J.G., Barnett J.A. (1991)： Passage of molecules through yeast cell wall: a brief eassay-review. Yeast 7:313-323

Eliyahu, D., Michalovitz, D., Eliyahu, S., Pinhasi-Kimhi, O.,and Oren,M. (1989)： Wild-type p53 can inhibit oncogene-mediated focus formation.Proc.Natl.Acad.Sci.USA. 86:8763-8767

Eldeiry W.S. (1998)： Regulation of p53 downsterm genes.Semin.
Cancer Biol.8:345-357

Folkers G.E., Buuren V. and Kaptein R. (2004)：Expression screening, protein purification and NMR analysis of human protein domains for structural genomics. Journal of Structural and Functional Genomics 5: 119–131
Funk W.D., Pak D.T., Karas R.H., Wright W.E., and Shay
J.W. (1992)： A Transcriptionally Active DNA-Binding Site for Human p53 Protein Complexes. MOLECULAR AND CELLULAR BIOLOGY 12, 2866-2871

Hinnen A., Meyhack B., Heim J. (1989)： Heterologous gene expression in yeast. Yeast Genetic Engineering. pp.193-214

Hitzeman R.A., Hagie F.E., Levine H.L., Goeddel D.V., Ammerer G., Hall B.D. (1981) Expression of a human gene for interferon in yeast. Nature 293:717- 722

Jenkins J.R., Rudge K., Chumakov P., and Currie G.A. (1985)： The cellular oncogene p53 can be activated by mutagenesis.Nature 317:816-818

Jenkins J.R., Rudge K., Chumakov P., and Currie G.A. (1984)： Cellular immortalization by a cDNA clone encoding the transformation-associated phosphoroprotein p53.Nature.312:651-654

Janson J.C., Rydén L.L. (1998) ： Protein Purification : Principles, High Resolution Methods, and Applications. Jon Wiley & Sons, New York, NY.

Lasser, A.B., Buskin J. N., Lockshon D., David R.L., Apone S., Hauschka S.D., and Weintraub H. (1989)：MyoD is a sequence-specific DNA binding protein requiring a myc homology to bind to the muscle creatine kinase enhancer. Cell 58:823-831
Lane D.P., and Crawford L.V. (1979)：T antigen is bound to a host protein in SV40-transformed cells.Nature 278:261-263

Lauren S., and Kathleen J.D. (1990)： Method to identify genpmic targets of DNA binding proteins. Proc. Natl. Acad. Sci. USA . Vol.87, 3274-3278

Lukyanov K.A., Gurskaya N.G., Bogdanova E.A., and Lukyanov S.A. Selective Suppression of Polymerase Chain Reaction . Russian Joumal of Bioorganic Chemisty, Vol. 25. No. 3/999:141-147

Natio M., Agata K., Otsuka K., Kino K., Ohta M., Hirose K., Perry M. M., and Eguchi G. (1991)： Embryonic expression of beta-actin lacZ hybrid gene injected into the fertilized ovum of the domestic fowl. Int. J. Dev. Biol. 35:69-75

Phaff H.J. (1990)： Isolation of yeast from natural source, In: Labeda DP(ed) isolation of biotechnological organisms from nature. MC Graw-Hill, New York, pp.53-79

Roy P., and Richard T. (1990)： A sensitive method for the determination of protein-DNA binding specificities. Nucleic Acids Research Vol.18, No.21: 6197-6204

Simith R.A., Duncan M.J., Moir D.T. (1985)： Hererologous protein secretion from yeast. Science 229: 1219-1224

Shuster J.R. (1989)： Regulated transcriptional systems for the production of proteins in yeast: regulation by Carbon source. Yeast Genetic Engineering pp.83-108

Scopes R.K. (1993) ： Protein Purification : Principles and Practice, Third Edition ed. Springer-Verlag, New York, NY.

Sano M., Ohyama A., Takase K., Yamamoto M., and Machida M. (2001)： Electrophoretic Mobility Shift Scanning Using an Automated Infrared DNA Sequencer. Bio Techniques Vol. 31, No. 5

Uwe K., and Dietmar K. (1998)： A subtractive hybridisation method for the enrichment of moderately induced sequences. Nucleic Acids Research.Vol. ,1359-1361

Vidal M., Legrain P. (1999)： Yeast forward and reverse ‘n’-hybrid systems. Nucleic Acids Research 27,919-929

Veitch N.J., Maugeri D.A., Cazzulo J.J., Lindqvist Y., and Barrett M.P. (2004) ： Transketolase from Leishmania mexicana has a dual subcellular localization. Biochem. J., 382, 759–767

Valenzuela P., Medina A., Rutter W.J., Ammerer G., Hall B.D. (1982) ：
Synthesis and assembly of hepatitis B virus surface antigen particles in yeast. Nature 298:341-350

Wright W.E., M.Binder, and W.Funk. (1991)： Cyclic Amplification and Selection of Targets(CASTing)for the Myogenin Consensus Binding Site .Molecular and Cellular Biology, Aug.4104-4110
Zsebo K.M., Lu H.S., Fieschko J.C., Goldstein L., Davis J., Duker K., Suggs S.U., Lai P.H., Bitter G.A. (1986)： Protein secreattion from Saccharomyces cerevisiae directed by the prepro-a-factor leader region. J Biol Chem 261:5858-5865