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研究生(外文):Ridha Handriany Danata
論文名稱(外文):Transgenic zebrafish eggs containing hepcidin as a novel feed supplement enhances fish immunity against pathogen infection
指導教授(外文):Shao-Yang Hu
口試委員(外文):Jue-Liang HsuYenny Risjani
外文關鍵詞:Antimicrobial peptideHepcidinTransgenicEmbryoGene Expression
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病原體引起的疾病問題往往造成水產養殖嚴重損失,大多數魚類疾病是由細菌性病源體所引起。抗生素通常被使用於治療處理水產養殖疾病問題,然而抗生素的使用可能導致環境污染問題,以及藥物殘留和抗藥性致病菌散佈而影響人體健康。抗菌胜肽(antimicrobial peptide; AMP)是宿主先天免疫的重要防禦組成。AMP會損壞或干擾病原體的細胞膜,並調節免疫抵抗病原體感染。鐵調素(Hepcidin)是一種小的生物活性肽,具有作為抗菌胜肽和平衡體內鐵的主要激素調節劑的雙重作用。本研究中將鐵調素hepcidin 基因與EGFP以斑馬魚卵母細胞特異性ZP3啟動子表現,並用於建立Tg(Zp3:hepc)轉基因斑馬魚。透過RT-PCR和西方墨點法證實F2受精卵中有重組Hepc與EGFP蛋白的表達。實驗結果顯示,餵食表達鐵調素受精卵3個月的成年斑馬魚與對照組相比,免疫反應如IL-1β, IL-6, IL-10, IL-15, TNF-α, COX-2, NF-ĸB, C3b, and TLR-4α具有較高之表現量,此結果說明表現鐵調素之受精卵可作為飼料補充物於應用於增強免疫抵抗疾病的感染
Pathogens induced disease cause significant stock losses in aquaculture. Most of the fish disease are caused by bacterial pathogens. Antibiotics are commonly applied to treat the disease in aquaculture, however the use of antibiotics for aquatic animals may not only lead environmental pollution problems but also can affect human health due to drug residues and the development of resistant strains of pathogens. Antimicrobial peptides (AMPs) are an important component of host defense. AMPs can damage or interfere with the cell membranes of pathogens and regulate immune responses against pathogens infection. Hepcidin is a small bioactive peptide with dual roles as an antimicrobial peptide (AMP) and as the principal hormonal regulator of iron homeostasis. In the present study, an expression plasmid in which hepcidin gene fused with EGFP was driven by zebrafish oocyte-specific ZP3 promoter and used to establish Tg (Zp3:hepc) transgenic zebrafish. The expression of recombinant hepc-EGFP protein in F2 fertilized eggs were confirmed by RT-PCR and Western blot. The result shown adult zebrafish fed with hepc-expressing fertilized eggs for 3 months exhibited higher expression level of immune response genes such as IL-1β, IL-6, IL-10, IL-15, TNF-α, COX-2, NF-ĸB, C3b, and TLR-4α compared to those in control group suggesting hepc-expressing fertilized eggs used as diet supplementation can enhance immunity against disease infection.
Chinnese abstract i
English abstract .ii
Acknowledment iv
Table of Contents v
List of Figures vii
List of Tables .viii
List of Appendix ...ix
Chapter I. Introduction 1
1.1 Background 1
1.2 Purpose of Study 3
Chapter II. Literature Review .4
2.1 Antimicrobial Peptide 4
2.2 Hepcidin 5
2.3 Zebrafish 7
2.4 Fish Immune System 9
Chapter III. Materials and Methods 13
3.1 Animals 13
3.2 Chemicals 13
3.3 Facilities and Equipments 15
3.4 Construction Plasmid Design 17
3.5 Microinjection and TEEA Analysis 18
3.6 Zebrafish Transgenic Line 18
3.7 Embryo Production 18
3.8 Genomic DNA Extraction 19

3.9 RNA Extraction 19
3.10 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 20
3.11 Polymerase Chain Reaction (PCR) 20
3.12 Gel Electrophoresis 21
3.13 Protein Extraction 21
3.14 SDS-PAGE 22
3.15 Western Blot 24
3.16 Detecting Immune-Related Gene Expression 25
3.17 Stastistical Analysis 25
Chapter IV. Result 26
4.1 Construction Plasmid Design 26
4.2 Transient Embryonic Excision Assay (TEEA) 29
4.3 Screening of Zebrafish Transgenic by F0 Tg (ZP3:AN-hepc:ZP3:EGFP) 29
4.4Germ-line transmission 33
4.5 Effect of feed supplement cointaining AN-hepc for immune-related gene 36
Chapter V. Discussion 39
Chapter VI. Conclusion ..41
References ..42
Supplementary Data ..48
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