臺灣博碩士論文加值系統

腸病毒71型屬於ss(+) RNA 病毒，Picornaviridae病毒科，是引起手足口症、泡疹性咽峽炎的病原之一。1998年在台灣發生大規模的流行造成405例的重病個案，其中有78個案例因神經性或心肺性併發症而死亡。腸病毒71型感染對象多半是三歲以下的小孩，目前主要疫區為亞太地區。為防治腸病毒71型，接種疫苗被認為是最有效的手段，但疫苗仍處在研發或臨床試驗階段。另外研發抗體作為初期感染的偵測或診斷也可以防治腸病毒71型的傳播。透過基因工程的方式可以在噬菌體上表現不同型式的抗體。在本篇研究中，主要是透過biopanning的方式從噬菌體人類單鏈抗體庫中篩選可以對抗腸病毒71型的片段抗體。利用限制性片段長度多型性分析(RFLP)將385個隨機挑選的clones分成5群。從中分別選擇5個clone做定序，並將定序的結果在IMGT的資料庫中比對抗體序列。發現5個clone都帶有抗體重鏈變異區(VH)，但只有clone 3及clone 9有抗體輕鏈變異區。雖然clone 3及clone 9在基因分析上表現完整的單鏈抗體的序列，但在西方點墨法中並沒有看到有單鏈抗體蛋白質的表現。儘管如此，在另外的clone 6及13表現儘管只重鏈變異區的片段中，發現這兩個clone對腸病毒71型具有結合能力，不管是在ELISA、西方點墨法或是免疫螢光實驗都可以証實。在本篇的研究中，我們從噬菌體人類單鏈抗體庫中篩選出兩個有潛力的噬菌體人類抗體株可以應用於腸病毒的偵測或研究上。

Enterovirus 71 (EV71) which causes hand-foot-mouth disease (HFMD) and herpangina belongs to the Picornaviridae family with ss(+) RNA virus. In 1998, EV71 outbreak in Taiwan and resulted in 78 fatal cases of 405 severe complications related neurological or cardiopulmonary symptoms. EV71 has been threatened the children under 3 years of age in Asia-Pacific area. Vaccination of EV71 seems the most efficient to eradicate the infectious disease. However, the development of vaccines is still in clinical trials. Antibodies also develop for early detection and diagnosis to control and prevent EV71 transmission. Different antibody format can be expressed by phage display technology via genetic engineering. Here, we selected the phage clones to against EV71 from the human scFv antibody library via biopanning. Procedure of biopanning included binding the target, washing out unbound phage and amplification of binders. Phage clones in total 385 were random picked and grouped by RFLP with SalI restriction enzyme. Five groups were identified and sequenced the nucleotides. Sequence data were aligned with the international ImMunoGeneTics information system (IMGT). We found that all of five phage clones contain a variable heavy sequence. Phage clone 3 and 9 contained full length scFv nucleotide sequence but formed no functional scFv antibody in protein level. However, the phage clones 6 and 13 showed binding ability to against EV71 that revealed in ELISA results. In addition, the phage clone 6 and 13 can recognize EV71virus particles and VP2 protein in western blot. Phage clone 6 and 13 detected EV71 in RD cell can be observed by immunofluorescence assay (IFA). In this study, the data suggested that phage clone 6 and 13 from the human scFv antibody library can be applied for EV71 research or diagnosis.

Contents
摘要................................................... I
Abstract............................................... II
致謝................................................... III
Contents............................................... IV
List of Figures.........................................VI
List of Table...........................................VII
Abbreviations...........................................VIII
Introduction............................................1
Enterovirus 71 (EV71)...................................1
Epidemiology and pathogenesis...........................1
Virology of EV71........................................1
Control and prevention..................................2
Antibody phage display..................................3
Overview................................................3
Antibody................................................4
Structure of the M13 bacteriophage......................5
Phagemid vector.........................................5
Construction of scFv phage display......................6
Experimental design.....................................7
Materials and Methods...................................8
Materials...............................................8
Single chain fragment variable (scFv) human phage library.................................................8
Bacteria strain and M13KO7 helper phage.................8
Cell, Virus and VP2 protein.............................8
Methods.................................................9
Rescue phage library....................................9
Biopanning for anti-EV71 phage clones...................9
Titering of phage and phage quantification by ELISA.....11
Identify the phage clones...............................11
Phage ELISA.............................................12
Western blot............................................12
Immunofluorescence assay (IFA)..........................13
Results.................................................14
Selection of phage clones which bind to EV71 by biopanning..............................................14
Grouping and sequencing analysis of selected phage clones..................................................14
Phage ELISA for MP4 strain and VP2 protein of EV71......15
Phage clones expressed the pIII protein.................15
Detection of EV71 by phage Western blot.................16
Determination of EV71 proteins by silver staining of SDS PAGE....................................................17
Characterization of phage clones by immunofluorescence assay...................................................17
Discussion..............................................18
Improve selecting a high affinity phage by using competitor in biopanning...........................................18
Human variable heavy and light chains were identified in phage clones by IMGT....................................18
Recognition of EV71 viral proteins by phage clones......19
Phage clones applied for immunofluorescence assay.......20
Conclusion..............................................21
References..............................................34
Appendix I. M13 phage structure.........................38
Appendix Π. Antibody phage display library construction39
Appendix III. Expected EV71 viral proteins..............40
Appendix IV.............................................41
1. Materials............................................41
1-1. Chemicals..........................................41
1-2. Antibodies.........................................42
1-3. Instruments........................................42
2. Buffer, Reagents and Kit.............................44
2-1. Phage Rescue.......................................44
2-2. PCR................................................45
2-3. Plasmid extraction.................................45
2-4. ELISA..............................................45
2-5. Protein electrophoresis............................46
2-6. Western blot.......................................47
2-7. Sliver staining: ProteoSilver Silver Stain Kit (PROT-SIL2, Sigma, USA).................................47

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