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研究生:王廷先
研究生(外文):Ting-Hsien Wang
論文名稱:應用多重SELEX資料庫篩選適體於高靈敏度免疫型qPCR禽流感H5感測之研究
論文名稱(外文):Ultra-sensitive Immuno-qPCR based Avian Influenza H5 Detection using Aptamers Selected from Multiple SELEX libraries
指導教授:陳林祈
指導教授(外文):Lin-Chi Chen
口試委員:劉嚞睿周崇熙陳倩瑜
口試日期:2014-07-04
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生物產業機電工程學研究所
學門:工程學門
學類:機械工程學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:93
中文關鍵詞:核酸適體微珠載体系統配位子指數增益演繹程序H5 血球凝集素免疫型qPCR皮莫耳量級親和力異位結合
外文關鍵詞:aptamermicro-beads based SELEXH5 hemagglutininimmuno-qPCRpico-molar affinityavidity
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經由系統配位子指數增益演繹程序(SELEX)所篩選出對目標蛋白分子具有專 一性辨識能力之核酸適體在流感病毒的快速篩檢上具有相當的潛力。然而,如何在 短時間及少量的蛋白質樣品消耗下找到高專一性與親合性的核酸適體序列一直是 核酸適體篩選研究中所遇到的瓶頸。本篇論文中,我們提出了一種利用微珠做為固定載體的有效率的核酸適體篩選方法。在此方法下,我們使用三種具有不同引子的 單股 DNA 資料庫對重組之 H5 血球凝集素進行核酸適體的篩選。其結果顯示,此 微珠載体之核酸適體序列篩選方式可有效的在早期的篩選迴圈(<4)便得到高度親 合性(~nM)且高度收斂(>90%)的單股 DNA 資料庫。在所篩選出的候選核酸適體序 列中,序列 ISP14 對 H5 血球凝集素具有卓越的親和力(45 pM),且經驗證後發現,ISP14 利用免疫型聚合酶連鎖反應(immuno-qPCR)方式在人類血清蛋白的干擾環境 下對 H5 血球凝集素的偵測極限(LOD)可達約 10-9 莫耳濃度,對於臨床的流感病毒偵測可為具有相當的潛力。此外,電腦模擬的分子嵌合結果中指出,此法所篩選出 之核酸適體序列與 H5 血球凝集素的結合位不盡相同,代表很有機會從此批核酸適 體篩結果中找出具有異位結合特性之核酸適體序列以提升流感病毒感測的靈敏度。

Aptamers, single-stranded nucleotides selected through Systematic Evolution of Ligands by EXponential enrichment (SELEX) that recognize its target protein specifically, are now considered promising in rapid diagnosis of influenza virus. However, identifying high affinity and specificity aptamer sequences in short time at low sample consumption have always been the bottleneck of SELEX works. In this thesis, an efficient micro-beads based SELEX was demonstrated with 3 distinctly primered ssDNA libraries in screening of H5 ultra-sensing aptamers by using recombinant H5 hemagglutinin (HA) as the selecting target. The result indicated that the micro-beads based SELEX was capable of obtaining ssDNA pools with low dissociation constant (~nM) and high convergence (>90%) in very early round of selection (<4). Among the selected aptamer candidates, ISP14 possessed outstanding affinity (45 pM) to the HA and was also validated being able to perform immuno-qPCR based HA detection with nano-molar ranged LOD in presence of interfering HSA, which showed great potential in clinical influenza virus detection. Furthermore, the in silico docking result provided evidences that selected aptamers bind to different epitopes on HA, implying chance of finding bivalent aptamers with avidity effect, which may dramatically improve the sensitivity of influenza virus detection.

Contents
Abstract........................................................................................................................... i
Chinese Abstract............................................................................................................. ii
Content........................................................................................................................... iii
List of Figures................................................................................................................. v
List of Tables................................................................................................................. viii
Chapter 1 Introduction .................................................................................................... .. 1
1.1. Research background ....................................................................................... .. 1
1.2. Objectives of the thesis .................................................................................... ...2
1.3. Strategy of this thesis ....................................................................................... .. 2
Chapter 2 Literature Review........................................................................................... .. 5
2.1. Aptamer and SELEX ....................................................................................... .. 5
2.2. In silico structure prediction and molecular docking ...................................... 11
2.3. Primer issue in aptamer selection .................................................................... 13
2.4. Affinity and specificity assay .......................................................................... 14
2.4.1 Separation based techniques .................................................................. 16
2.4.2 Mixture based techniques ...................................................................... 19
2.5. Influenza A and avian flu detection ................................................................. 23
Chapter 3 Material and Methods .................................................................................... 28
3.1. Apparatus and reagents .................................................................................... 28
3.2. Micro-beads based SELEX of anti-H5 aptamer .............................................. 33
3.2.1. ssDNA libraries design ......................................................................... 33
iii
3.2.2. Preparation of epoxide-functionalized glass microbeads (EGBs) ........ 34
3.2.3. Screening of antagonistic aptamer against recombinant H5
hemagglutinin ................................................................................................. 34
3.2.4. Sequencing and sequence alignment .................................................... 37
3.3. Identifying potential bivalent aptamer via in silico docking method .............. 42
3.4. Immuno-qPCR based affinity assay................................................................. 43
3.5. Limit of detection and specificity assay .......................................................... 47
Chapter 4 Result and Discussion .................................................................................... 49
4.1. The evolution of H5-binding ssDNA ligands monitored by differential qPCR
analysis ................................................................................................................... 49
4.2. Affinity improvement of the ssDNA pools during anti-H5 SELEX ............... 60
4.3. In silico analysis of selected aptamer candidates ............................................ 63
4.3.1. Multiple sequence alignment and clustering ........................................ 63
4.3.2. Molecular docking with hemagglutinin ................................................ 67
4.4. Promising anti-H5 aptamer with superior affinity ........................................... 74
4.5. Nano-molar level hemagglutinin detection with immuno-qPCR method ....... 77
Chapter 5 Conclusions .................................................................................................... 80
Reference ........................................................................................................................ 82
Appendix ........................................................................................................................ 88
iv

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