臺灣博碩士論文加值系統

神經壞死病毒(NNV)在水產養殖上為一重要的魚類病毒，尤其在石斑魚的養殖上更是造成嚴重的經濟損失。NNV屬於結病毒科(Nodaviridae)的betanodavirus屬，神經壞死病毒的病毒顆粒大小在25∼30微米左右，無封套膜、二十面體，屬於核糖核酸病毒，具有兩段單股的正股RNA，稱為RNA1與RNA2，RNA1所產生的蛋白質為RNA 聚合酶；而RNA2所產生的蛋白質為鞘蛋白(capsid protein)，主要是組成病毒外殼。石斑魚從卵孵化到吋苗的這段期間內，遭受NNV病毒感染的機率極大，被感染的魚苗會有不正常如旋轉般的游動情況，稱為飛旋症；感染後並會引起魚的神經壞死(viral nervous necrosis, VNN)，腦脊髓炎(encephalomyelitis)和液泡形成、腦病和視網膜病變(vacuolating encephalopathy and retinopathy, VER)，並造成大量的死亡，死亡率甚至會高達90~100％也是近年來在石斑魚養殖上所面臨最嚴重的病毒性疾病。
核糖核酸干擾(RNAi)技術在近幾年來被詳細的探討與應用，對於抑制基因的表現上被視為一重要的工具。本實驗我們預計利用核糖核酸干擾技術發展抗NNV病毒的水產疫苗，而基於疫苗作用的持續時間與成本上的考量，我們將嘗試以DNA載體為基礎的siRNA來發展疫苗。經由建立的siRNA穩定表現魚體的品系，做病毒感染的結果顯示，比較控制組，其F1子代能擁有較高的存活比率。原位雜交的結果更是顯示siRNA的表現載體，能在魚體內成功的產生siRNA的片段，這將讓我們更具信心將此技術應用於發展石斑魚抗NNV病毒的疫苗上。
而本實驗另一項重要的目的便是探討利用微脂粒(nano-liposome)作為傳遞系統來發展疫苗的可行性。我們利用六種不同配方之微酯粒(nano-liposome)包裹能表現紅螢光的siRNA之構築體，混合魚類飼料(孵化之豐年蝦)，經由投餵方式讓一個月大之斑馬魚(zebrafish)食入，接著觀察其在魚體內表現之情形。確定其食入後16小時，將魚體置於螢光顯微鏡下觀察，發現斑馬魚腹部有很強之紅螢光表現。 接著對魚體做各個組織分離，抽取RNA後做RT-PCR，結果顯示，腦，心臟，眼睛，腸，甚至肌肉都能偵測到螢光基因的表現，而免疫組織染色也得到類似的結果。表示利用微酯粒包覆混合飼料餵食法，能夠成功的將基因帶入生物體內，進而使其能有效的在體內表現。這樣的結果我們預測利用如此的傳遞系統對未來應用於腸道基因轉殖或是疫苗的製造將有其發展的潛力。

Viral infections have been increasingly reported in cultured marine fish. Among these infections, viral encephalopathy and retinopathy (VER), caused by betanodaviruses (piscine nodavirus), is one of the most devastating, occurring in a variety of marine fish all over the world. Disease control would evolve an important issue for the whole world aquatic industry. Histological examination of tissues from the central nervous system and the retina often reveals areas of conspicuous tissue vacuolation and necrosis. Their genome consists of two molecules of messenger sense RNA. RNA1 is approximately 3.1 kb in size and carries the gene that encodes for an RNA-dependent RNA polymerase referred to as protein A; RNA2 is approximately 1.4 kb in size and contains the open reading frame that encodes the capsid protein.
Short interfering RNAs (siRNAs) have proved to be a useful tool in studying gene function. Recently, small interfering RNAs (siRNAs) have been used for gene knockdown in mammalian cultured cells, but their utility in fish has remained unexplored. In this thesis study, we want to development anti-fish virus by using RNAi technology, and nano-liposome was the delivery system that we used in this experiment.
We constructed 4 sets of siRNA vectors containing NNV''s RNA2 gene in the position at 93, 585, 730 and 1024 nt. We also constructed the plasmids with HcRed gene regarding as siRNA detection marker. My experiment is separated into two parts: one is using siRNA expression vector inject zebrafish to single out stable line, and the F2 generation was challenge by NNV. There is higher survival rate compared to control. In the F1 generation, we could detect fluorescence marker gene in expression vector by RT-PCR. The other part：we used nano-liposome as DNA deliver tool with oral administration in zebrafish. Six different formulations of liposome were examined for the fluorescent marker gene of siRNA expression vector . The zebrafish was fed with nona-liposome, 4 h to 12 h after feeding, and the samples were collected at every interval of 2 h of this period. At 16 h after liposome feeding, there was strong fluorescence expressing in several organs including heart, eye and intestine etc. The result of in situ hybridization demonstrated vector-mediated siRNA could be expressed in zebrafishes. Therefore, vector-based siRNA delivered by nano-liposome has the potential to be the vaccine that against aquatic virus.in aquaculture.

Content
English abstract………………………………………………………...i
Chinese abstract……………………………………………………….iii

Introduction…………………………………………………………1
Materials .........................……………………………………………8
A. Biotic materials……………………………………………………..8
1. Zebrafish (Danio rerio)………………………………………...8
2. Virus……………………………………………………………8
3. Competent cell………………………………………………….9
4. Plasmids………………………………………………………...9
5. The formulation of lipoplex…………………………………….9
B. Equipments and instruments……………………………………….10
1. High speed centrifuge…………………………………………..10
2. Centrifuge t……………………………………………………...10
3. Electrophoresis equipment……………………………………...10 4. PCR system………………………………………………….….10
5. Spectrophotometer………………………………………….......10
6. Fluorescent microscope…………………………………………10
7. Glassmicroelectrode puller……………………………………...10
8. Microinjector……………………………………………………10
9. Microgrinder…………………………………………………….10
10. Polytron…………………………………………………………10
11. pH meter………………………………………………………...10
12. CO2 water jacketed incubator…………………………………...10
13. Vortex Mixer…………………………………………………….10
C. Reaction reagents…………………………………………………...10
1. Bacterial medium………………………………………………..11
2. Enzymes........................................................................................12
3. Molecular weight marker………………………………………..12
D. Biotic Reaction Reagent Kits……………………………………….13
1. QIAquick® Gel Extraction Kit (QIAGEN)……………………...13
2. PureLinkTM HiPure Plasmid DNA Purification Kits (Invitrogen).13
3. SuperScriptTM III First-Strand Synthesis System for RT-PCR
(Invitrogen)………………………………………………………13
4. RNA-spinTM Total RNA Extraction Kit 【For cell/tissue】…….13
5. Platinum® Taq DNA polymerase (Invitrogen)…………………...13
6. SuperScriptTM III One-Step RT-PCR System with Platinum® Taq DNA polymerase (Invitrogen)…………………………………..13
7. PureLinkTM HiPure Plasmid Maxiprep Kits (Invitrogen)………..13
E. Reaction solution and buffer…………………………………………13
1. DNA electrophoresis and buffers………………………………...13
2. RNA electrophoresis and buffers………………………………...14
3. Plasmid boiling prep. - Extraction buffer ( Lysis buffer )………..14
4. PEG solution for competent cell (Calcium Chloride solution)…..15
5. In Situ Hybridization buffers……………………………………..15
F. Medium/Buffer for Cell Culture……………………………………...17
Cell culture medium………………………………………………...17
G. Cell culture Drugs……………………………………………………28
H. Chemical Drugs ……………………………………………………..19
1. Tris-base, glacial acetic acid, NaCl, Glucose, potassium acetate, EDTA, HCl, NaOH, Chloroform, Formamide, Formaldehyde, Ethidium bromid (EtBr), Ampicillin, ammonium persulfate, Methanol. (Merk)………………………………………………...19
2. Saturated phenol for DNA (AMRESCO)………………………...19
3. Bacto-agar, yeast extract, Bacto-trypton (DIFCO)……………….19
4. SeaKem® LE agarose (CAMBRREX)…………………………...19
5. SeaKem® GTG agarose (CAMBRREX)…………………………19
6. TRIZOL® reagent (Invitrogen)…………………………………...19
7. Phenol：chloroform：isoamyl alcohol (25：24：1) (Sigma)……19
8. Chloroform：isoamyl alcohol (24：1) (Sigma)………………….19
I. Pimers....................................................................................................19
J. RNA probe (labeled with DIG-UTP)…………………………………19
Methods
A. plasmid DNA extraction……………………………………………..20
B. Microinjection of zebrafish embryos………………………………..20
C. Virus challenge………………………………………………………20
D. Liposome oral administration……………………………………….21
E. HcRed immunohistochemical staining……………………………....21
F. Tissue distribution and RNA extraction……………………………...21
G. Reverse transcription-polymerase chain reaction (RT-PCR).………..22
H. Whole-mount In Situ Hybridization…………………………………22

Result
A. dsRNA design and Construction of shRNAs expression plasmid…..24
B. In situ hybridization analysis of the expression of siRNA in the zebrafish…………………………………………………………..…25
C. Selection of siRNA expression stable line and virus challenge in
F1 generation………………………………………………………..25
D. Characterisation of the liposome and lipoplex formulations..……….25
E. Detection of HcRed reporter RNA in several tissues
of lipoplex-feeding zebrafish by RT-PCR…………………………..26
F. Immunohistochemical staining…………………………………..…..26
G. The time - course analysis of lipoplex-feeding fishs………………...27
Discussion..............................................................................................28
References.............................................................................................31












Index of Tables
Table 1. Anti-NNV siRNAs target selection……………………………40
Table 2. RPS (relative percent survival)………………………………...41
Table 3. Liposome deliver efficiency test……………………………….42
Table 4. Liposome 2 deliver efficiency test……………………………..43













Index of Figures
Figure 1. SiRNA inhibited NNV replication in vitro experiment ……...44
Figure 2. Vector-based siRNA expression construct………………......45
Figure 3. In situ hybridization analysis of the expression of siRNA in the zebrafish………………………………………………………...46
Figure 4. Transfecting siRNA expression vector into eggs of fish by microinjection…………………………………………………...47
Figure 5. The result of RT-PCR of F1 generation of siRNA injected fishes…………………………………………………………….48
Figure 6. Liposome-feeding fishes tissue distribution and RT-PCR…...49
Figure 7. Immunohistochemistry of liposome- feeding fish…………...50
Figure 8. Time course of liposome delivery system in zebrafish……....51
Figure 9. Time course analysis of liposome-feeding fish by RT-PCR....52











Index of Appendix
Appendix I. Light microscopy of tissue sections from turbot. Sections from a turbot 16 days post-hatching immunolabelled for nodavirus (SJNNV-antiserum)………………………………………………53
Appendix II. The structure of Nodavirus (Flock House Virus)………....54
Appendix III. Full-length nucleotide and deduced amino acid sequences of GGNNV RNA1……………………………………………….55
Appendix IV. Full-length nucleotide and deduced amino acid sequences of GGNNV RNA2……………………………………………….56
Appendix V. A model for the mechanism of RNAi…………………….57

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