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研究生:許琬婷
研究生(外文):Wan-Ting Hsu
論文名稱:以SELEX技術篩選蛋白質分子的DNA核酸適體-篩選程序評估
論文名稱(外文):Sreening DNA aptamers of protein molecules using SELEXtechnique-Evaluation of screening protocols
指導教授:曾銘仁
指導教授(外文):Min-Jen Tseng
口試委員:李政怡謝文馨周禮君
口試委員(外文):Cheng-I LeeW. H. HsiehChau, Lai-Kwan
口試日期:2011-01-26
學位類別:碩士
校院名稱:國立中正大學
系所名稱:分子生物研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2011
畢業學年度:99
語文別:中文
論文頁數:99
中文關鍵詞:蘭花病毒核酸適體腫瘤壞死因子
外文關鍵詞:SELEXaptamer
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用來偵測蘭花病毒齒舌蘭輪斑病毒(ORSV)與蕙蘭嵌紋病毒(CymMV)和腫瘤壞死因
子α(TNF-α)的技術主要是酵素結合免疫吸附法(ELISA),雖然ELISA 方便偵測,卻
不易用於快速或大量樣品的偵測。近年的研究指出,在in vitro 下使用SELEX 技術
可以篩選與標的分子專一結合的核酸適體(aptamers),核酸適體具有抗體的優點,
也具更好的穩定度和容易合成。
本論文應用SELEX 方法,使用純化的GST-CymMV CP 和GST-ORSV CP 融合蛋白和
64mer 的隨機ssDNA library 作13 輪的篩選,因蛋白品質不佳導致無法篩選出具有
高度專一性與結合力的aptamer。另外純化的GST- TNFα和64mer 的隨機ssDNA
library 作11 輪的篩選,將篩選的PCR 產物接到yT&A 載體,確定後加以定序。利
用Mfold 軟體預測每一個序列的二級結構,可分類成三種不同的二級結構構型。進
而再根據序列和構形歸類出兩組相似的aptamers,進一步做親合力測試,然而所挑
選的aptamers 不具高的專一性和親合力。
為了改進aptamers 的專一性,對篩選的條件作改善與評估,調整目標蛋白與ssDNA
library 的比例,將目標蛋白的分子數減少和減短與ssDNA 結合的時間、及尋找篩
選buffer 的Mg2+濃度、測定每一輪PCR 產物的ssDNA 分子數、重新設計ssDNA library
兩端的序列使之不互補、也將GST 融合蛋白換成His 融合蛋白降低篩選時的影響。
根據改善的條件,使用純化的TNFα-His6 和83 mer 的隨機ssDNA library 作10 輪
的篩選,定序篩選到的PCR 產物結果顯示ssDNA library 污染到先前實驗使用的
64mer 的隨機ssDNA library。實驗的過程務必使用更嚴謹的篩選條件且時常更換
PCR 的reagents 才能有效的找到在結構中含有保留序列的aptamers。最後篩選出的
aptamer,可利用化學合成法生成,以探討物理性的結構及用在生物上的測試。
The major technique for detecting orchid virus, ORSV and CymMV, and TNFα,
an inflammatory mediator, is ELISA. Though ELISA is an efficient method,
it is hard to apply for quick and high-throughput onside detection. Recently,
in an in vitro test tube system, the SELEX technology was applied to screen
for single-stand nucleic acid aptamers which can interact with target
molecule with high specificity and high affinity. The aptamers contain not
only the advantages of antibodies, but also present better stability and easy
to synthesized by chemical methods.
This study applied purified GST-CymMV-CP and GST-ORSV-CP to screen
aptamers from a 64 mer random ssDNA library using SELEX for 13 cycles. We
failed to obtain aptamers with high specificity and high affinity due to the
poor quality of proteins. The purified GST-TNFα was also gone through the
same SELEX screen for 11 cycles. The obtained PCR products were cloned into
yT&A plasmids and the inserts were sequenced. The secondary structures of
every DNA sequence were predicted by online Mfold software, they can be
clustered into three different structure classes. Two groups of similar
aptamers were selected for further affinity test according their sequences
and structures. However, they didn’t show good specificity.
In order to improve the specificity of aptamers, we evaluated and
modified the screening protocols as follow. Readjust the ratio of target
protein and ssDNA library. Lower the molecule numbers of target protein and
shorten the interacting time between target protein and ssDNA library. Test
for the optimal Mg2+ concentrations in screening buffer. Determine the
molecule numbers of ssDNA after each screening cycle. Redesign the fixed
sequences flanking the random sequences of ssDNA library to avoid
complementary annealing between them. Substitute GST-fusion protein to a
much smaller His-tagged protein to prevent the influence of GST during
screening.
Using the modified protocol, the purified TNFα-His6 was used to screen
aptamers from an 83 mer random ssDNA library 10 cycles. The DNA sequences
of obtained aptamers showed that there was contamination of previous 64 mer
random ssDNA library. To obtain specific aptamers with high affinity, a more
stringent screening procedure must be follow and often make fresh PCR
reagents. Finally, the optimal aptamers can be synthesized with chemical
method to analyze physical structures and apply as biological detection
probes.
英文摘要
中文摘要
目次
第一章、 前言
1-1 Systematic Evolution of Ligands by Exponential
Enrichments( SELEX)………………………………………………………………1
1-2 核酸適體(Aptamer)…………………………………………………………………2
1-3 核酸適體(Aptamer)與抗體的比較…………………………………………………3
1-4蘭花病毒………………………………………………………………………………4
1-4-1蕙蘭嵌紋病毒(Cymbidium mosaic virus, CymMV)
1-4-2 齒舌蘭輪斑病毒(Odontoglossum ringspot virus,ORSV)………………5
1-4-3 目前偵測蘭花病毒的方法
1-5 腫瘤壞死因子α(TNF-α).…………………………………………………………6
1-6 實驗目的…………………………………………………………………………….7
第二章、實驗材料與方法
第一節、DNA 的操作
1-1 質體之建構
1-1-1 pGEX/CymMV 和pGEX/ORSV 鞘蛋白質體之建構………………………………8
1-1-2 pET-28a/TNFα質體之建構
1-2 瓊脂凝膠電泳………………………………………………………………………9
1-3 以電泳在透析膜中回收純化瓊脂膠內的DNA(DNA electro-elution)…………10
1-4 接合反應(Ligation)………………………………………………………………10
1-5 製備勝任細胞(Competent cell)…………………………………………………11
1-5-1 氯化銣法(Rubidium Chloride method)製備勝任細胞(Competent cell)
1-5-2 製備E.coli strain BL21(DE3)CodonPlus competent cell……………13
1-5-3 電穿孔法 (electroporation) 製備勝任細胞 (competent cell)…… 13
1-6 轉形作用 (Transformation)
1-6-1 Yeastern Biotech 轉型作用 (ECOSTM101)…………………………………14
1-6-2 化學性轉型作用 (Chemical Transformation)………………………… 15
1-6-3電穿孔轉型作用(Transformation by Electroporation)-
Bio-Rad Gene Pulser….........................................16
1-7 質體之製備 (Plasmid Preparation)
1-7-1 微量質體粗萃取- LiCl-Phenol-chloroform extraction method............16
1-7-2 組套純化微量 (mini)質體(BioKit).............................17
1-7-3 組套純化微量 (midi)質體(NucleBond PC100 kit).................17
1-8 限制酶鑑定 (Restriction enzyme digestion)..........................19
1-9 凍菌保存..........................................................19
1-10 DNA 定序.........................................................19
第二節、基因重組蛋白質的操作
2-1 誘導目標蛋白質在大腸桿菌的表現....................................20
2-1-1(a) 小量誘導GST融合蛋白表現
2-1-1(b) 小量誘導His6-tag融合蛋白表現
2-1-2 大量誘導蛋白質表現以及萃取蛋白質……………………………………21
2-2 分析目標蛋白質是否形成不溶的inclusion body………………………………22
2-3 蛋白質定量…………………………………………………………………………23
2-4 SDS 膠體電泳法( Electrophoresis)…………………………………………… 24
2-4-1 SDS-PAGE (SDS-Polyacrylamid Gel Electrophoresis)的製備
2-4-2 SDS-PAGE 的染色及褪染………………………………………………… 25
2-5 西方墨點分析法 (Western-blotting analysis)………………………………26
2-6 GST 融合蛋白的大量表現與純化……………………………………………… 29
2-6-1(a) GST-TNFα 融合蛋白的大量表現
2-6-1(b) GST-CymMV 和GST-ORSV 鞘蛋白融合蛋白的大量表現………………29
2-6-2 GST融合蛋白的純化……………………………………………………… 30
2-7 His6-tag 融合蛋白的大量表現……………………………………………………31
2-7-1 His6-tag融合蛋白的大量表現
2-7-2 His6-Tag融合蛋白的純化
2-8 SELEX 方法 (Systematic Evolution of Ligands by …………………………33
EXponential Enrichment)
2-8-1 目標蛋白與DNA 第一輪的結合篩選
2-8-2 PCR 方法擴增篩選的DNA……………………………………………………34
2-8-3 利用streptavidin 與biotin 的高親合力結合以
取得單股DNA……………………………………………………………… 36
2-8-4 Semiquantitive PCR 方法判別每一輪篩選ssDNA…………………………37
2-8-5 GST 融合蛋白的counter selection………………………………………38
2-8-6 定序與預測aptamer 二級結構…………………………………………… 38
2-8-7 目標蛋白與aptamers 的affinity 測試………………………………… 39
第三章、實驗結果與討論
3-1 針對GST-cymMV鞘蛋白和GST-ORSV鞘蛋白(CP)融合蛋白……………………….41
3-1-1 純化以IPTG 誘導大量表現的GST-cymMV 鞘蛋白和GST-ORSV 鞘蛋白(CP)
融合蛋白
3-1-2 以SELEX 篩選結合GST-cymMV CP 和GST-ORSV CP 融合蛋白的aptamers..41
3-1-3 GST-cymMV CP 和GST-ORSV CP 融合蛋白經SELEX 篩選後無法得到結合力
較佳的aptamers……………………………………………………………42
3-1-4 GST-cymMV CP 和GST-ORSV CP 融合蛋白與ssDNA library 結合時間
與量的檢討………………………………………………………………….43
3-1-5 SELEX 過程使用的selection buffer 的影響…………………………….43
3-1-6 利用GST-cymMV CP融合蛋白重新做SELEX篩選…………………………..43
3-2 針對GST-TNFα融合蛋白………………………………………………………….44
3-2-1 純化以IPTG誘導大量表現的GST-TNFα融合蛋白………………………..44
3-2-2 GST-TNFα融合蛋白與ssDNA library 的SELEX 結果…………………..45
3-2-3 整理與歸納aptamer 的定序結果………………………………………….47
3-2-4 預測與分類aptamer 的二級結構………………………………………….47
3-2-5 測試aptamer 與GST-TNFα融合蛋白的親合力……………………………48
3-2-6 GST-TNFα融合蛋白經由SELEX 篩選出的aptamers 無較高的結合力….48
3-2-7 GST 蛋白部份會影響GST-TNFα融合蛋白與ssDNA 的結合……………..49
3-2-8 DNA library 兩端與PCR 引子互補的固定序列的設計與長度…………..50
3-2-9 GST-TNFα融合蛋白與ssDNA 分子結合時間與量的調整………………..50
3-2-10 SsDNA 是否須先自行折疊再與GST-TNFα融合蛋白進行SELEX 篩選….51
3-3 針對His-TNFα融合蛋白………………………………………………………….51
3-3-1 經由IPTG 誘導表現與純化獲得His-TNFα融合蛋白…………………….51
3-3-2 TNFα-His6 蛋白與ssDNA 進行SELEX 篩選………………………………..52
3-3-3 整理與歸納aptamer 的定序結果………………………………………….53
3-3-4 預測aptamer 的二級結構………………………………………………….53
3-3-5 TNFα-His6 融合蛋白經由SELEX 篩選無得到專一的aptamers………….54
第四章、實驗總結…………………………………………………………55
圖表目錄
表1. 實驗中PCR 使用的引子序列…………………………………………………….56
表.2 共72 個長度為64mer aptamers 定序結果…………………………………76-77
表.3 共21 個長度為64mer aptamers、7 個長度為83mer aptamers 定序結果….90
圖1.SELEX 流程………………………………………………………………………….2
圖2. CymMV CP 和ORSV CP 定序序列………………………………………………..57
圖3.TNFα 定序序列…………………………………………………………………….58
圖4. GST-CymMV CP 融合蛋白的大量表現及純化…………………………………..59
圖5. GST-ORSV CP 融合蛋白的大量表現及純化…………………………………….60
圖.6 以西方墨點法確定GST-CymMV CP 融合蛋白和GST-ORSV CP 融合蛋白………61
圖.7 GST-CymMV CP 和GST-ORSV CP 融合蛋白與ssDNA library 第一輪
的SELEX 篩選…………………………………………………………………….62
圖.8 GST-CymMV CP 和GST-ORSV CP 融合蛋白與ssDNA library 的第二輪
的SELEX 篩選…………………………………………………………………….63
圖.9 GST-CymMV CP 和GST-ORSV CP 融合蛋白經由SELEX 篩選13 輪的曲線圖……64
圖.10 GST-CymMV CP 融合蛋白與ssDNA library 第一輪的SELEX 篩選……………65
圖.11 GST-CymMV CP 融合蛋白與ssDNA library 的第二輪的SELEX 篩選…………66
圖.12 GST-CymMV CP 融合蛋白與ssDNA library 的第二輪的SELEX 篩選…………67
圖.13 使用GST 蛋白做連續counter selection 三次SELEX 篩選結果…………..68
圖14. Glutathione 磁珠上的GST-CymMV CP 和GST-ORSV CP 融合蛋白確認…….69
圖15. GST-TNFα 融合蛋白的大量表現及純化……………………………………….70
圖.16 GST-TNFα 融合蛋白與ssDNA library 第一輪的SELEX 篩選………………..71
圖.17 GST-TNFα 融合蛋白與ssDNA library 的第二輪的SELEX 篩選……………..72
圖.18 GST-TNFα 融合蛋白與ssDNA library 的第十一輪的SELEX 篩選…………..73
圖.19 GST-TNFα 融合蛋白經由SELEX 篩選11 輪的曲線圖………………………….74
圖.20 使用GST 蛋白做counter selection 的結果…………………………………75
圖.21 利用AlignX程式比對72個序列的結果…………………………………………78
圖.22 Mfold 預測aptamer 的二級結構…………………………………………..79-80
圖.23 Mfold 預測aptamer 的二級結構……………………………………………….81
圖.24 Mfold 預測aptamer 的二級結構……………………………………………..82
圖.25 兩組序列有高度相同且結構相似的aptamers…………………………………83
圖.26 Aptamer 的affinity 能力測試………………………………………………..84
圖27. TNFα-His6 融合蛋白的大量表現及純化……………………………………….85
圖.28 以西方墨點法確定TNFα-His6融合蛋白……………………………………….86
圖.29 TNFα-His6 融合蛋白與ssDNA library 第一輪的SELEX 篩選……………….87
圖.30 TNFα-His6 融合蛋白與ssDNA library 的第二輪的SELEX 篩選…………….88
圖.31 TNFα-His6 融合蛋白與ssDNA library 的第十輪的SELEX 篩選…………….89
圖.32 利用AlignX 程式比對28 個序列的結果……………………………………..91
圖.33 Mfold 預測aptamer 的二級結構……………………………………………….92
參考文獻……………………………………………………………….93-99
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