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研究生：	李雪鈴 Li, Syue-Ling
論文名稱：	篩選結合登革病毒第二型NS5 DNA核酸適體做為檢測及治療用途 Identification of DNA Aptamers Targeting NS5 protein of type 2 Dengue Viruses for diagnostic and/or therapeutic purpose
指導教授：	王憲威 Wang, Shainn-Wei
學位類別：	碩士 Master
系所名稱：	醫學院 - 分子醫學研究所 Institute of Molecular Medicine
論文出版年：	2019
畢業學年度：	107
語文別：	英文
論文頁數：	61
中文關鍵詞：	登革病毒、去氧核酸適體、NS5蛋白、系統性配體演化指數擴增法、次世代定序分析、解離常數、酵素連結免疫吸附分析法、核酸連結免疫吸附分析法
外文關鍵詞：	Dengue virus NS5, DNA aptamer, SELEX, NGS, Dissociation constant, ELISA, ELONA
相關次數：	點閱：55 下載：3
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登革病毒(DENV)乃經病媒蚊傳染，廣泛傳染流行於熱帶及亞熱帶區域包含東南亞、南美洲、西太平洋地區，特別好發於雨季過後，且近年來更似有向全球各地蔓延的趨勢。感染登革病毒患者一般會出現輕微發燒類似感冒症狀或嚴重者出現登革出血熱的症狀，尤其現也發現有不少的案例是屬於無症狀，惟迄今仍無針對DENV的藥物或疫苗來防治登革病毒感染。DENV屬於黃熱病毒科，含四種血清型(DV1-4)，其基因是正股RNA，可轉譯成一條多胜肽蛋白由三個結構蛋白(C-M-E)以及七個非結構蛋白(NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5)組成。其中的NS5蛋白具有甲基化轉移酶(MTase)與RNA依賴性RNA聚合酶(RdRp)的功能。MTase作用在病毒RNA的5’端，致使其甲基化後類似宿主的RNA結構，以利於逃脫宿主免疫反應；而RdRp則負責產生大量病毒RNA，誠然NS5蛋白在病毒生活史中扮演重要的角色。另外，NS5的蛋白質序列保守性在登革四型中高於其他病毒蛋白，若以它為DENV偵測或藥物結合的評定目標應是極佳的選擇。基於此，我們利用系統性配體演化指數擴增法(SELEX)篩選出一些具有專一結合力的短片段核酸適體(Aptamer)，來作為DENV感測探針，甚且進行治療登革病毒抑制劑之可能性探討。為此，本研究(1)以細菌質體(pET15b)為載體建構帶有組胺酸標籤的NS5 重組蛋白(6xHis-NS5)並確認了具功能的6xHis-NS5，再(2)導入純化後的6xHis-NS5蛋白於SELEX，經由15輪的篩選終能得到該專一性的適體，並藉次世代定序分析定序之，結果取得了三個DNA序列相似的適體族群，繼之(3)從其中各挑出一個出現頻率最高者(稱為A1、A2、A3)當作具有潛力之適體候選者，遂以ELISA的技術評比其對病毒NS5的親和效果，發現A3親和力最高，A2最低(A3>A1>A2，其解離常數(Kd)分別為22, 32, and 12 nM。最後(4)利用ELONA做為初步測試作為感測探針之功能，結果顯示此三個候選適體，非但皆可對於第二型登革熱感染之細胞所產生的NS5蛋白，而且登革熱一至四型的6xHis-NS5重組蛋白也都可被偵測到。另外，在抑制登革病毒複製的效果方面，從目前的結果尚未能發現較有效的DNA適體。總言之，此研究提供了數個對登革病毒NS5蛋白有專一結合力的適體當作潛力性的偵測試劑，未來尚需在病毒繁衍的抑制性或干擾性治療上，有更深入的探討，盼選擇到最小且有效結合序列的適體，或尚需加入其他化學修飾以延長DNA適體在細胞內之半衰期等，期能達到限制病毒感染並避免產生有害的免疫反應之目的。

The mosquito-borne Dengue virus (DV) is a single positive-stranded RNA virus belonging to the family Flaviviridae. Four serotypes, DV1–4, have caused widespread epidemics in human and nearly 40% of the world’s population is at risk of being infected. Patients may suffer from life threatening dengue hemorrhagic fever (DHF) with or without shock (DSS) and present with wide spectrum of clinical manifestations. No vaccines and antivirals have been approved to treat DV so far. The DV NS5 is the viral RNA-dependent-RNA-polymerase, which is highly conserved among the four serotypes and therefore represents an effective target for diagnosis and/or antiviral therapeutics. To screen novel ligands for a desired molecular target, a SELEX (Systematic evolution of ligands by exponential enrichment) technology is employed as an in vitro selection method to evolve target-specific nucleic acid ligands, called aptamers. In comparison to target-specific antibodies, aptamers are less likely to induce host immune responses and thus a useful alternative for biomedical studies and disease therapy. In this study, we aimed to develop DV2 NS5-specific aptamers which are potent for diagnosis and prevention of pandemic DVs. Our results indicated a successful purification of recombinant NS5 with His tag at the N-terminus (6xHis-NS5) and the selection of 6xHis-NS5 specific aptamers through 15-rounds of SELEX. Sequence identification of cycling enriched aptamers by NGS revealed three major groups of homologous aptamer sequences in cycle 10 to 15. Three dominant aptamer candidates of each group called A1, A2 and A3 were identified and capable of pulldown His-NS5. The binding affinity of these biotinylated aptamer was in the order of A3>A1>A2, by ELISA assay, with the Kd of 22, 32, and 12 nM, respectively. Most importantly, Aptamer-based ELONA assay, established as a diagnostic tool with detection limits in the range of 40-1000 ng of His-NS5, was capable of specific detection of NS5 from lysates of DV2-infected cells. However, inhibition of DV2 replication in Huh7 cells was not achieved with the three aptamer candidates during intracellular interaction, presumably due to short half-live of three aptamer candidates. This study identified three promising aptamers to be used for diagnostic purpose. Further dissecting of these aptamers to identify structural domains with specific binding and/or functional domains may be required to improve their applications.

摘要	II
Abstract	III
Acknowledgements	IV
Index	V
Figure Index	VII
Table Index	VII
Supplementary Figure Index	VIII
Supplementary Tables Index	VIII
Introduction	1
Epidemic of Dengue Fever	1
Serotypes and Genome Organization of Dengue Virus	1
Diagnostic Tools for Dengue Virus	2
Therapies of Dengue Virus Infectoin	3
Aptamer and Systematic Evolution of Ligands by Exponential Enrichment (SELEX)	3
Aptamer Applications in Biosensor	4
Aptamer Applications in Therapeutics	5
Goals and specific aims:	6
Materials and Methods	7
Cells and virus	7
Plaque assay	7
Reagents and Antibodies	8
Plasmids and constructs:	11
Recombine protein expression and purification	11
SDS-PAGE gel staining and Western blot analysis	12
Systematic evolution of ligands by exponential enrichment (SELEX)	13
Pull-down assays	14
Binding affinity measurements of aptamer candidates by ELISA	15
ELONA (Enzyme- Linked Oligonucleotide Assays) development for viral detection	16
Aptamer transfection for functional assays and half-life determination	16
Functional inhibition of viral replication	17
Confocal imaging analyses of subcellular aptamers and their half-life	17
Cell viability assay	18
Results	19
Induction and expression of the 6xHis-NS5 recombinant protein	19
Purification of the recombinant 6xHis-NS5 of DV2	19
Screening of 6xHis-NS5-specific aptamers by Systematic Evolution of Ligands by EXponential Enrichment (SELEX).	20
Next generation sequencing (NGS) analysis of cycling-eluted apatmers	21
Three candidate aptamers A1-3 captured recombinant His-NS5 with high affinities	23
Aptamer-based ELONA assay for specific and sensitive detection of Dengue NS5 from DV2-infected cells	23
Inhibition of DV2 replication in Huh7 cells was not achieved with the 3 aptamer candidates during intracellular interaction.	24
Discussion	26
References	33
Figures	37
Tables	50
Supplementary Figure	53
Supplementary Tables	54


Figure Index

Fig. 1	The gene maps of plasmids pET28a-DV2-NS5 and pET-15b-DV2-NS5 ................. 37
Fig. 2	Induction and expression of the DV2 6xHis-NS5 recombinant protein ………...…. 38
Fig. 3	Purification of the DV2 6xHis-NS5 recombinant protein …………………….….... 39
Fig. 4	The preparation of DNA Aptamer library and its step-wise evolution through Systematic Evolution of Ligands by Exponential Enrichment (SELEX) ………….. 41
Fig. 5	Ex-situ quantification and verification of cycling enriched DV2 6xHis-NS5 specific aptamers after SELEX …………………………………….……………………….. 42
Fig. 6	Binding specificities and dissociation constants of three aptamer candidates to recombinant DV2 6xHis-NS5 …………………….……………………………..… 43
Fig. 7	Enzyme Linked Oligonucleotide assay (ELONA) development for DV2 NS5 detection ………………………………………………………………….…….….. 45
Fig. 8	Effects of aptamer candidates to DV2 replication and cell viability. (A) Time course evaluation of DV2 NS5 production during viral infection ….……………………... 47
Fig. 9	The decay of Aptamers after transfection into Huh7 cells ………………..……...… 49
Table Index
Table 1	Ranking and grouping of highly occurrent aptamer sequences between SELEX cycles 10 to 15 ……………………………………………………………………………... 51
Table 2	The percent occurrence and the representative Weblogo sequences of the three conserved aptamer populations in the cycles of C10 to C15 ………………………………...…. 52
Table 3	Sequences and secondary structures of three selected aptamer candidates …………. 5 
Supplementary Figure Index
Fig. S1	Imaging analyses of the decay of Aptamers A2 and A3 ………………………….… 54






Supplementary Tables Index
Table S1	Summary of pre-processed NGS data of cycling enriched aptamers ………………... 55
Table S2	Top 50 hit sequences of cycle-10 enriched aptamers ……………………………...… 56
Table S3	Top 50 hit sequences of cycle-11 enriched aptamers ………………………………... 57
Table S4	Top 50 hit sequences of cycle-12 enriched aptamers ………………………………... 58
Table S5	Top 50 hit sequences of cycle-13 enriched aptamers ………………………………... 59
Table S6	Top 50 hit sequences of cycle-14 enriched aptamers ……………………………...… 60
Table S7	Top 50 hit sequences of cycle-15 enriched aptamers ………………………………... 61
                                    

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校內：2024-02-01公開
校外：2024-02-01公開

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