臺灣博碩士論文加值系統

哺乳類動物的細胞具有脂質雙層膜作為保護細胞的天然屏障，因此外來的分子要穿越細胞膜進入細胞中非常困難。過去Stephen Dowdy團隊自TAT全長蛋白質中找到一段由鹼性胺基酸所組成之蛋白轉導區(protein transduction domain; PTD)，稱之為TAT-PTD。這種PTD胜肽具有跨越細胞膜之能力，因此Matsushita等人認為將此PTD應用於攜帶基因進入細胞中便可克服細胞膜的屏障。而常見的具有PTD之轉位蛋白，除了上述提到之TAT-PTD之外，還有VP22-PTD。本論文構築了含有TAT及VP22之VP22-Delta重組蛋白質，其中的Delta [(SPKR)4 -iTAT-CspA]是仿效美國專利PAT. NO. 6835810製備而成。並另外於VP22之C端接入一DNA-binding protein TmHU而構築出VP22-TmHU重組蛋白質。分別將VP22-Delta及VP22-TmHU依不同之比例與pEGFP-N1 DNA共同作用後加入培養中之細胞。結果顯示各重組蛋白與DNA結合後，並無攜帶DNA進入細胞之能力。此外，在重組蛋白質VP22-TmHU的C端連結一綠色螢光蛋白構築出VP22-TmHU-GFP重組蛋白質，經細胞結合試驗，證實VP22-TmHU並不具有進入細胞之能力。VP22-Delta及VP22-TmHU雖無直接攜帶DNA轉染細胞的能力，但若與商品化之微脂粒Lipofectamine併用，則顯著提高微脂粒轉殖效率。因此這些重組蛋白質可降低微脂粒Lipofectamine的使用量。將VP22-TmHU的C端額外接入一核定位訊號(nuclear localization signals, NLS)，構築而成VP22-TmHU-NLS，則該重組蛋白質經細胞轉導試驗證實具有攜帶DNA進入細胞之能力，因此該重組蛋白質將來可作為動物細胞轉殖DNA的輔助工具。

Molecules can not easily enter the cell because the cell membrane of the cell is composed of a phospholipid bi-layer, acting as a natural, protective barrier. In previous publication, Stephen Dowdy and researchers found a peptide within the TAT genome consisted of basic amino acid sequences called TAT-PTD (Protein Transduction Domain). Since PTD peptide can pass through the cell membrane barrier, Matsushita and researchers used it to carry genes into the cell. Common translocatory proteins containing the PTD include TAT-PTD and VP22-PTD. In this thesis, recombinant proteins of VP22-Delta, a construct combining VP22-PTD, and Delta/(TAT) [(SPKR)4-iTAT-CspA] (according to US Patent No. 6835810), were expressed in E. coli prokaryotic expression system. Furthermore, recombinant proteins of a fusion protein, VP22-TmHU, combining VP22-PTD and to its C-terminus, TmHU, a DNA-binding protein, were expressed. Subsequently, different ratios of VP22-Delta and/or VP22-TmHU to pEGFP-N1 DNA were mixed and transfected into the cells. Transfection efficiencies were observed and calculated by flow cytometry. Results showed that the 2 different recombinant proteins were not able to deliver DNA into the cell. In addition, VP22-TmHU-GFP, a recombinant protein of VP22-TmHU fuses with GFP, was constructed, expressed, and used in a cell membrane association assay. Again, results showed that VP22-TmHU-GFP was not carried into the cell upon trasnfection. However, when VP22-TmHU and/or VP22-Delta were used in conjunction with commercialized liposome (Lipofectamine) in transfection, transfection efficiency was significantly higher than Lipofectamine alone. Thus these recombinant proteins greatly minimize the amount of lioposome (Lipofectamine) needed for transfection. Additionally, when expressed recombinant proteins of VP22-TmHU-NLS, a construct of VP22-TmHU and a nuclear localization signal sequence fused to its C-terminus, were used in a transduction assay, results showed that VP22-TmHU-NLS was a successful nucleotide-transducting protein. Therefore, it is a useful tool to aid the delivery of transgenic DNA into animal cells.

目錄
頁
中文摘要..........................................................................................................................i
英文摘要.........................................................................................................................ii
目錄................................................................................................................................iii
表目錄............................................................................................................................vi
圖目錄...........................................................................................................................vii
第一章 緒言..................................................................................................................1
第二章 文獻探討..........................................................................................................3
第一節、 動物體的基因轉殖...................................................................................3
第二節、 蛋白轉導技術(Protein Transduction Technology) ..................................4
第三節、 轉位蛋白(translocatory protein)簡述.......................................................5
第四節、 轉導機制...................................................................................................6
第五節、 DNA binding protein.................................................................................7
第六節、 增進基因傳遞之研究概況.......................................................................8
實驗策略一.....................................................................................................................9
實驗策略二...................................................................................................................10
實驗策略三...................................................................................................................11
第三章 材料與方法....................................................................................................12
第一節、質體DNA之構築......................................................................................12
1. VP22的序列合成及TA cloning.................................................................12
2. 質體DNA之製備.......................................................................................13
3. PCR法增幅特定DNA片段.......................................................................14
4. PCR產物確認及純化.................................................................................14
5. 限制酶切割反應(Restriction Enzyme Cleavage Reaction).......................15
6. 接合反應(Ligation)....................................................................................15
7. 勝任細胞(Competent cell)的製備.............................................................16
8. 轉形作用(Transformation).........................................................................16
9. 重組質體的挑選與確認............................................................................16
10. 重組質體的序列......................................................................................17
第二節、基因重組蛋白的表現................................................................................17
1.重組蛋白表現..............................................................................................17
2. 十二硫酸脂鈉-多聚丙烯醯胺膠體電泳分析
(Sodium dodecyl sulfate polyacrulamide gel electrophoresis, SDS-PAGE)...18
3. 西方墨漬法確認重組蛋白表現................................................................18
第三節、基因重組蛋白之純化.................................................................................19
1. 可溶態重組蛋白純化................................................................................19
2. 不可溶態（包涵體）重組蛋白之純化........................................................20
3. 蛋白質定量................................................................................................21
第四節、重組蛋白質之特性分析..............................................................................21
1. 重組蛋白質與DNA結合能力測試...........................................................21
1.1. 質體pEGFP-N1之大量製備.............................................................21
1.2. 洋菜膠電泳延阻試驗(Electrophoresis Mobility Shift Assay, EMSA) ..............................................................................................22
2.重組蛋白質與DNA結合之定性分析.........................................................22
3. 純化之重組蛋白質轉染之效率................................................................23
4. 以流式細胞儀分析重組蛋白質轉染之效率............................................25
5. 以SAS進行統計分析................................................................................25
6. 以VP22-TmHU-GFP為工具探討VP22-TmHU對細胞之作用..............25
第四章 結果................................................................................................................27
第一節、各基因重組蛋白之選殖............................................................................27
1. pTmHU-pET24a、pTmHU-pET44a及pDelta-pET44a之次選殖.........27
2. VP22的序列合成及TA cloning...........................................................27
3. pVP22-TmHU-pET24a及pVP22-Delta-pET24a之構築.....................27
4. pVP22-TmHU-GFP-pET24a之構築....................................................27
5. pVP22-TmHU-NLS-pET24a之構築...................................................27
第二節、 各基因重組蛋白之表現與純化.............................................................28
1. TmHU及Delta.........................................................................................28
2. VP22-TmHU、VP22-Delta、VP22-TmHU-NLS及VP22-TmHU-GFP...28
第三節、重組蛋白質之特性分析............................................................................29
1. 純化之重組蛋白質與DNA結合能力測試............................................29
2. 純化之重組蛋白質與DNA結合之定性分析........................................29
3. 純化之重組蛋白質轉染DNA之效率....................................................29
4. 以流式細胞儀分析螢光細胞數目.........................................................30
5. VP22-TmHU-GFP與細胞結合的情形....................................................31
第五章 討論................................................................................................................32
附錄...............................................................................................................................38
參考文獻.......................................................................................................................91

表目錄
頁
表一、轉位蛋白之特性.................................................................................................38
表二、轉位蛋白之胺基酸及其極端鹼性之特性.........................................................38
表三、合成VP22之特異性引子序列...........................................................................49
表四、增幅TmHU、Delta及VP22基因之特異性引子序列表...................................51
表五、增幅GFP及NLS DNA合成的引子序列表......................................................52
表六、SDS-PAGE膠片之配方......................................................................................53
表七、各濃度之VP22-TmHU併用不同量lipofectamine之轉染結果...........................81
表八、各濃度之VP22-Delta併用不同量lipofectamine之轉染結果.............................81
表九、各濃度之VP22-TmHU-NLS併用不同量lipofectamine之轉染結果..................84
圖目錄
頁
圖一、以PTD作為攜帶各種巨分的傳遞工具，可多方面應用..................................39
圖二、蛋白轉導之可能機制.........................................................................................40
圖三、仿效美國專利PAT. NO. 6835810所合成之Delta序列....................................41
圖四、仿效美國專利PAT. NO. 6835810所合成之Delta示意圖................................41
圖五、質體pTmHU-pET24a及pTmHU-pET44a的構築............................................42
圖六、質體pDelta-pET44a的構築...............................................................................43
圖七、質體pVP22-TmHU-pET24a的構築..................................................................44
圖八、質體pVP22-Delta-pET24a的構築.....................................................................45
圖九、質體pVP22-TmHU-GFP-pET24a的構築..........................................................46
圖十、質體pVP22-TmHU-NLS-pET24a的構築........................................................ 47
圖十一、以引子延伸法合成之VP22 DNA序列圖.....................................................48
圖十二、以四段引子合成VP22之示意圖...................................................................49
圖十三、pET system 原核表現系統............................................................................50
圖十四、以PCR法增幅TmHU基因之洋菜膠電泳分析圖........................................54
圖十五、以PCR法增幅Delta基因之洋菜膠電泳分析圖...........................................54
圖十六、TmHU選殖入原核表現載體pET24a之電泳分析圖....................................55
圖十七、Delta選殖入原核表現載體pET44a之電泳分析圖......................................55
圖十八、將TmHU選殖入pET24a及pET44a後進行DNA定序之結果....................56
圖十九、將Delta選殖入pET24a及pET44a後進行DNA定序之結果......................56
圖二十、以PCR法增幅VP22之洋菜膠電泳分析圖..................................................57
圖二十一、VP22序列分析圖.......................................................................................58
圖二十二、VP22選殖入pTmHU-pET24a重組質體之電泳分析圖...........................59
圖二十三、Delta選殖入pVP22-pET24a重組質體之電泳分析圖..............................59
圖二十四、VP22-TmHU序列分析圖...........................................................................60
圖二十五、VP22-Delta序列分析圖.............................................................................61
圖二十六、GFP選殖入pVP22-TmHU-pET24a重組質體之電泳分析圖..................62
圖二十七、pVP22-TmHU-GFP-pET24a序列分析圖..................................................63
圖二十八、以PCR法增幅NLS基因之洋菜膠電泳分析圖........................................64
圖二十九、NLS選殖入pVP22-TmHU-pET24a重組質體之電泳分析圖..................64
圖三十、VP22-TmHU-NLS序列分析圖......................................................................65
圖三十一、以28℃誘導TmHU/pET44a/BL21 codon及
TmHU/pET44a/BL21 DE3重組蛋白質表現之SDS-PAGE分析圖.......66
圖三十二、以28℃誘導Delta/pET44a/BL21 Codon重組蛋白質表現
之SDS-PAGE分析圖..............................................................................67
圖三十三、以SDS-PAGE及Western法分析重組蛋白TmHU及Delta示意圖….....68
圖三十四、以37℃誘導VP22-TmHU/pET24a/BL21 Codon
重組蛋白質表現之SDS-PAGE示意圖....................................................69
圖三十五、以37℃誘導VP22-Delta/pET24a/BL21 Codon
重組蛋白質表現之SDS-PAGE示意圖....................................................70
圖三十六、以37℃誘導VP22-TmHU-GFP/pET24a/BL21 DE3
重組蛋白質表現之SDS-PAGE示意圖....................................................71
圖三十七、以37℃誘導VP22-TmHU-NLS/pET24a/BL21 Codon
重組蛋白質表現之SDS-PAGE示意圖....................................................72
圖三十八、以SDS-PAGE及Western法分析重組蛋白
VP22-TmHU及VP22- Delta示意圖........................................................73
圖三十九、以SDS-PAGE及Western法分析重組蛋白
VP22-TmHU-GFP示意圖.........................................................................74
圖四十、以SDS-PAGE及Western法分析重組蛋白
VP22-TmHU-NLS示意圖...........................................................................75
圖四十一、以EMSA法分析純化之重組蛋白質TmHU
及Delta與DNA結合能力之洋菜膠電泳分析圖....................................76

圖四十二、以EMSA法分析純化之重組蛋白質VP22-TmHU
與DNA結合能力之洋菜膠電泳分析圖..................................................77
圖四十三、以EMSA法分析純化之重組蛋白質VP22-Delta
與DNA結合能力之洋菜膠電泳分析圖..................................................78
圖四十四、以EMSA法分析純化之重組蛋白質VP22-TmHU-NLS及VP22-TmHU-GFP與DNA結合能力之洋菜膠電泳分析圖...................79
圖四十五、以Western blot分析VP22-Delta/24a/codon與DNA結合之能力............80
圖四十六、VP22-TmHU併用lipofectamine轉染CHO細胞之結果圖......................82
圖四十七、VP22-Delta併用lipofectamine轉染CHO細胞之結果圖.........................83
圖四十八、VP22-TmHU-NLS轉染CHO細胞之結果圖............................................85
圖四十九、以流式細胞儀分析VP22-TmHU及VP22-Delta
併用不同量的lipofectamine的轉染效率分析圖.....................................86
圖五十、以流式細胞儀分析VP22-TmHU及VP22-Delta與
lipofectamine、pFGFP-N1不同添加次序的轉染結果分析圖....................87
圖五十一、以流式細胞儀分析VP22-TmHU-NLS轉染CHO細胞的效率分析圖....88
圖五十二、以流式細胞儀分析VP22-TmHU-NLS與lipofectamine、
pFGFP-N1不同添加次序的轉染結果分析圖.........................................88
圖五十三、VP22-TmHU-GFP與CHO細胞結合分析圖.............................................89
圖五十四、以不同清洗的條件，測試VP22-TmHU-GFP
與 CHO細胞作用之分析圖.....................................................................90

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