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研究生中文姓名:薛嵐狄
研究生英文姓名:Randee Sheppard
中文論文名稱:CpG寡去氧核醣核酸對於點帶石斑魚抗神經壞死病毒口服疫苗之免疫調節功效
英文論文名稱:Modulatory effect of CpG oligodeoxynucleotide on an oral DNA vaccine against Nervous Necrosis Virus in orange-spotted grouper (Epinephelus coioides)
指導教授姓名:邱品文
呂明偉
口試委員中文姓名:助理教授︰林翰佑
教授︰陳歷歷
助理教授︰邱品文
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:水產養殖學系
學號:10433055
請選擇論文為:學術型
畢業年度:106
畢業學年度:105
學期:
語文別:英文
論文頁數:63
中文關鍵詞:點帶石斑神經壞死性病毒口服式DNA疫苗CpG寡去氧核醣核酸
英文關鍵字:Epinephelus coioidesnervous necrosis virusoral DNA vaccineCpG oligodexynucleotide
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本論文針對造成石斑魚幼苗大量死亡的主要病原神經壞死性病毒(NNV),成功地發展出一種有效的口服式DNA 疫苗。此外,也探討了使用類鐸受體9(TLR9)激活物CpG寡去氧核醣核酸(CpG ODN)作為此口服疫苗佐劑的功效及作用機制。實驗結果發現單獨使用此口服式DNA 疫苗進行免疫後即能在魚體內產生專一性抗體,顯示此疫苗能激發宿主的後天性體液性免疫反應。在免疫後14天時,在宿主頭腎內可見到TLR9、Mx及IL-1β 被誘發表現,意謂著TLR9訊息傳遞系統的確被啟動。實驗結果也顯示,在疫苗中添加CpG ODN佐劑能更進一步提升免疫後魚體內專一性抗體的力價。在攻毒試驗中,此口服式DNA 疫苗單獨使用時即能降低魚體內NNV 殼蛋白(capsid)基因的表現量;在疫苗內添加CpG ODN後能更進一步降低capsid 基因的表現量至與以肌肉注射疫苗組相當的程度。在攻毒2週後,添加CpG ODN的疫苗免疫組的專一性抗體力價顯著高於單獨疫苗免疫組,意謂著CpG ODN提升此口服DNA疫苗保護效力的機制可能與提升宿主體內的Th2免疫反應有關。據我們的瞭解,本論文為第一篇有關對石斑魚具有保護功效口服式DNA疫苗的研究報告
We report the development of an effective oral DNA vaccine pcMGNNV2 against nervous necrosis virus (NNV), a leading cause of mass mortality in grouper larvae. In addition, the modulatory effect of CpG oligodeoxynucleotide (ODN), a Toll-like receptor 9 agonist, on the oral DNA vaccine was evaluated. The DNA vaccine alone elicited the production of NNV-specific antibodies indicating that the vaccine was capable of triggering an adaptive humoral response. Furthermore, the induction of TLR9, Mx and IL-1β was observed in the head kidney on day 14 post-vaccination, supporting the claim that the vaccine could trigger a TLR9 signaling. The incorporation of CpG ODN further enhanced the production of NNV-specific antibodies. After challenging vaccinated fish with NNV, the vaccine alone decreased the level of NNV capsid gene expression. The combination of CpG ODN further decreased the expression of NNV to a level compatible to that of intramuscularly vaccinated fish. Interestingly, 2 weeks post viral infection, the oral vaccines appeared to induce a Th2 response as indicated by the higher antibody titer. The data suggested that CpG ODN augmented the oral vaccine, perhaps by enhancing the Th2 response. To our best knowledge, this is the first report of a successful oral DNA vaccine against NNV in grouper.
ACKNOWLEDGEMENTS i
摘要 ii
ABSTRACT iii
LIST OF FIGURES vii
CHAPTER 1: INTRODUCTION 1
CHAPTER 2: LITERATURE REVIEW 4
2.2 Orange-Spotted Grouper 4
2.2.1 Taxonomy 4
2.2.2 Morphology 4
2.2.3 Distribution and Life cycle 5
2.2.4 Market 6
2.2.5 Diseases 6
2.3. Nervous necrosis virus 7
2.3.1 Properties 8
2.3.2 Clinical sign 8
2.3.3 Prevention and control 8
2.3 Vaccination 10
2.4.1 Vaccination 10
2.4.1.1 Types of Vaccines 10
2.4.1.2 Live attenuated vaccines 10
2.4.1.3 Subunit vaccines 11
2.4.1.4 Toxoid Vaccines 11
2.4.1.5 Recombinant Vector Vaccines 11
2.4.1.6 DNA Vaccines 12
2.4.2 Vaccine Delivery 12
2.4.2.1 Immersion Vaccination 13
2.4.2.2 Injection Vaccination 13
2.4.2.3 Oral Vaccination 14
2.5. Adjuvant 15
2.5.1 Mineral Salts 15
2.5.2 Oil Emulsions 16
2.5.3 Particulate adjuvants 16
2.5.4 CpG Oligodeoxynucleotides 16
2.6 Fish Immunity 18
2.6.1 Lymphoid organs 18
2.6.1.1 Thymus 19
2.6.1.2 Spleen 19
2.6.1.3 Kidney 20
2.6.2 Innate immunity 21
2.6.2.1 Physical Barriers 21
2.6.2.3 Innate Cellular cytotoxicity 22
2.6.2.3.1 Complement 22
2.6.2.3.2 Interferon 23
2.6.2.3.3 Interleukins 23
2.6.2.3.4 Protease Inhibitors 24
2.6.2.3.5 Natural antibodies 24
2.6.3 Adaptive immunity 24
2.6.3.1 Antibodies 25
2.6.3.2 Cellular Cytotoxicity 25
2.6.3.3 Cytokines in adaptive immunity 26
CHAPTER 3: MATERIALS AND METHODS 27
3.1 Fish rearing 27
3.2 DNA vaccine and CpG ODN 27
3.3 Liposomes 27
3.4 Vaccination 28
3.5 Viral challenge 28
3.6 Measurement of specific antibody titer by enzyme-linked immunosorbent assay (ELISA) 29
3.7 Gene expression profiling 29
4.1. Expression of pcMGNNV2-encoded capsid protein in grouper cells 31
4.2 Protection efficacy of pcMGNNV2 oral vaccine with and without CpG ODN 31
4.3 Treatment effect of DNA oral vaccine and CpG ODN on production of specific antibodies 32
4.4 Effects of Oral DNA vaccine and CpG ODN on the expression TLR9 in head kidney of vaccinated fish 33
4.5 Expression of downstream genes of TLR9 in MX and IL-1β 34
4.6 Expression of T-cell markers in vaccinated fish 35
CHAPTER 5: DISCUSSION 37
CHAPTER 6: CONCLUSION 41
CHAPTER 7: REFFERENCES 42

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