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尼羅魚類toll受器18及其銜接分子之選殖與特徵鑑定

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Declaration IAcknowledgements IIAbstract IV中文摘要 VITable of Contents VIIList of Tables XIList of Figures XIIList of Abbreviations XVList of Publications XVIIChapter I: General Introduction 11.1. Aquaculture and Tilapia 21.2. Diseases of tilapia 21.3. P

attern Recognition Receptors 31.4. Toll-like receptors 41.5. Types, ligand specificity and sub-cellular localization of vertebrate TLRs 61.5.1. TLR1 family 81.5.2. TLR3 family 91.5.3. TLR4 family 101.5.4. TLR5 family 101.5.5. TLR7 family 111.5.6. TLR11

family 121.6. TLR signaling pathways 131.6.1. MyD88-dependent signaling pathway 131.6.2. TRIF-dependent signaling pathway 141.7. TLR accessory proteins 151.8. Current knowledge on the TLR1 family (including TLR18) 161.9. Significance and specific aims 17Chapte

r II: Fish-specific TLR 18 in Nile tilapia (Oreochromis niloticus) recruits MyD88 and TRIF to induce expression of effectors in NF-κB and IFN pathways in melanomacrophages 192.1. Abstract 202.2. Introduction 212.3. Materials and methods 222.3.1. Fish collection, immune sti

mulation, and cell culture 222.3.2. Cloning of full-length OnTLR18 and plasmid constructions 232.3.3. Bioinformatics 262.3.4. RNA isolation, cDNA synthesis, and quantitative real-time PCR 262.3.5. THK cells stimulation 282.3.6. Confocal microscopy 282.3.7. Examina

tion of effectors induced by TLR18 in THK cells 292.3.8. Coimmunoprecipitation and Western blotting 292.3.9. Statistical analysis 302.4. Results 302.4.1. Molecule cloning and in silico analyses 302.4.2. Quantitative analysis of basal expression pattern of OnTLR18 312

.4.3. Quantitative analysis of OnTLR18 expression level after S. agalactiae, A. hydrophila and poly I:C injection 322.4.4. Quantitative analysis of OnTLR18 expression level after TLR ligand stimulation 322.4.5. Subcellular localization of OnTLR18 322.4.6. Constitutively active f

orm of OnTLR18 promotes expression of cytokines, chemokines, type I IFNs and antimicrobial peptides 332.4.7. Physical interaction between OnTLR18 and adaptor proteins 332.5. Discussion 332.6. Conclusions 38 Chapter III: Functional characterization of myeloid differentiation f

actor 88 in Nile tilapia (Oreochromis niloticus) 593.1. Abstract 603.2. Introduction 613.3. Materials and methods 623.3.1. Sample collection 623.3.2. Total RNA extraction and cDNA synthesis 633.3.3. Cloning and bioinformatics analyses of OnMyD88 633.3.4. Pre

paration of expression plasmids 643.3.5. Confocal microscopy 643.3.6. Dual luciferase analysis 653.3.7. Overexpression of OnMyD88 in THK cells 653.3.8. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis 653.3.9. Immunoprecipitation (IP) and Western blot

ting (WB) analysis 653.3.10. Statistical analysis 663.4. Results 673.4.1. Cloning and sequence characterization 673.4.2. Phylogenetic analysis and genome synteny comparison 673.4.3. Domain organization 683.4.4. Tissue distribution of OnMyD88 in Nile tilapia 683

.4.5. Expression of OnMyD88 and cytokines in Streptococcus agalactiae challenged tilapia 683.4.6. Cellular localization of OnMyD88 683.4.7. Dual luciferase reporter assay 693.4.8. Cytokine gene induction in OnMyD88-overexpressing THK cells 693.4.9. Fish-specific TLR25 inte

raction of OnMyD88 693.5. Discussion 693.6. Conclusion 72 Chapter IV: Expression, signal transduction, and function analysis of TIRAP and TRIF in Nile tilapia (Oreochromis niloticus) 864.1. Abstract 874.2. Introduction 884.3. Materials and methods 904.3.1. I

n vivo sample collection 904.3.2. Extraction of total RNA and cDNA synthesis 914.3.3. Cloning of OnTIRAP and OnTRIF in Nile tilapia 914.3.4. Quantitative real-time PCR (qRT-PCR) analysis 924.3.5. Preparation of expression plasmids 924.3.6. Luciferase reporter assay 9

34.3.7. Confocal microscopy 934.3.8. Co-immunoprecipitation (Co-IP) and western blotting analysis 944.3.9. Statistical analysis 954.4. Results 964.4.1. Characterization of Nile tilapia TIRAP and TRIF 964.4.2. Evolutionary and genomic synteny analysis 964.4.3. D

omain organization 974.4.4. Expression patterns of OnTIRAP and OnTRIF in different tissues 984.4.5. OnTIRAP and OnTRIF mRNA expression levels in Nile tilapia after challenges 984.4.6. Cellular localization of OnTIRAP and OnTRIF 984.4.7. Activation of OnTIRAP and OnTRIF in sig

nal transduction 994.4.8. Interaction of OnTRIF with teleost TLR25 994.5. Discussion 1004.6. Conclusion 104Chapter V: General Discussion 123References 126