臺灣博碩士論文加值系統

本實驗室運用奈米金粒子作為高效率的疫苗載體，探討粒徑大小與抗體反應間的關係。首先將製備完成的口蹄疫病毒胜肽與禽流感病毒胜肽，與不同粒徑的奈米金（2奈米、5奈米、8奈米、12奈米、17奈米、37奈米至50奈米）做結合，為了確保能達到最大的鍵結量，因此我們在胜肽的酸基末端修飾一個半胱氨酸，強化鍵結能力。對照組方面，則以傳統的血氰蛋白與胜肽作結合之後，兩組接施打至老鼠體內去引發免疫反應。老鼠施打四周、六周、八周和十周後，開始採集血液，粒用免疫酵素檢測法，針對不同的胜肽與載體進行檢測。我們發現奈米金粒子在粒徑2奈米、5奈米、8奈米、12奈米和17奈米時，有良好的抗體反應。其最大的抗體反應量，其粒徑範圍是在8奈米至17奈米之間，與傳統的血氰蛋白的方式相比，其抗體反應量提高了將近三倍。值得注意的是，奈米金粒子載體本身並無檢測到任何抗體反應產生，但是血氰蛋白本身卻引起相當高的抗體反應，因此干擾了其所攜帶胜肽的抗體反應。此外，我們使用離子藕荷電漿分析儀計算分佈於老鼠脾臟中的奈米金粒子含量，我們發現此種免疫性質與不同粒徑的奈米金粒子在老鼠脾臟中的含量高度相關。在此研究中，我們發現奈米金粒子不但具有集中抗體辨識的功效，還可提高抗體反應的能力，並且奈米金粒子的粒徑大小與免疫行為有高度相關，其奈米金粒子粒徑範圍從8奈米至17奈米間，可當作最理想的胜肽疫苗載體。

To elicit the size-dependent carrier ability of gold nanoparticles (GNPs), synthetic peptide corresponding to foot-and-mouth disease virus (FMDV) and influenza A virus subtype (H5N1) viral proteins is conjugated to GNPs with diameter 2-nm, 5-nm, 8-nm, 12-nm, 17-nm, 37-nm, and 50-nm. Extra cysteine is added to the C-terminus of the FMDV peptide (pFMDV) and H5N1 peptide (pH5N1) to ensure maximum conjugation. Immunization of peptide-keyhole limpet hemocyanin (KLH) conjugate was performed as control. Blood are withdrawn on week 4, 6, 8, and 10. Titers against peptide and carriers are obtained. For peptide-GNP immunization, specific binding against peptide is detected in the sera of mice injected with 2-nm, 5-nm, 8-nm, 12-nm and 17-nm GNP conjugates. Maximum binding occur with GNPs of sizes between 8-nm to 17-nm. The peptide -GNPs induce 2.17 ~ 3 fold antibody responsecompared to peptide-KLH. In particular, all sera exhibited undetectable binding against GNP, while antisera of peptide-KLH present high levels of binding activity against KLH. Deposition of GNPs in mouse spleen was evaluated by ICP-MS. The immunogenicity of peptide is correlated to amounts of GNPs accumulated in spleen. In conclusion, we show the size-dependent immunogenic properties of peptide-GNP conjugates. GNPs ranging from 8-nm to 17-nm may serve as ideal carrier to elicit focused and enhanced antibody response against a synthetic peptide.

Contents
Acknowledgment---------------------------------------------I
Chinese Abstract------------------------------------------II
Abstract-------------------------------------------------III
Contents---------------------------------------------------V

Chapter 1
1.1 Introduction...........................................1
1.1.1 General Introduction to foot-and-mouth disease.......1
1.1.2 The outbreak of foot-and-mouth disease in Taiwan.....2
1.1.3 The structure of foot-and-mouth disease virus........3
1.1.4 Peptide Vaccine......................................5
1.1.5 The advantage of gold nanoparticle served as vaccine carrier....................................................5
1.1.6 Motivation...........................................6
1.2 Experiment and Method..................................7
1.2.1 Chemicals............................................7
1.2.2 Preparation and characterization of gold nanoparticle...............................................7
1.2.3 Designing of synthetic peptide.......................8
1.2.4 Preparation of pFMDV-carrier conjugates..............9
1.2.5 Immunization of mice.................................9
1.2.6 Enzyme-linked immunosorbent assay...................10
1.2.7 Gel-Electrophoresis of GNPs.........................11
1.2.8 Inductively coupled plasma mass spectrometry........11
1.2.9 Transmission electron microscopy....................12
1.2.10 Statistical Analyses...............................13
1.3 Result and Discussion.................................14
1.3.1 Preparation and analysis of pFMDV-GNP conjugates....15
1.3.2 The pFMDV-GNP conjugates induced focused and enhanced
antibody response.........................................17
1.3.3 Antibody response of pFMDV-GNP conjugates is associated with the amount of GNPs taken up by spleen.....22
1.4 Conclusions...........................................26
References................................................27

Chapter 2
2.1 Introduction..........................................30
2.1.1 General Introduction to Influenza A virus subtype H5N1......................................................30
2.1.2 The structure of Influenza A virus subtype H5N1.....31
2.1.3 Peptide Vaccine.....................................32
2.1.4 The advantage of gold nanoparticle served as vaccine carrier...................................................33
2.1.5 Motivation..........................................34
2.2 Experiment and Method.................................35
2.2.1 Chemicals...........................................35
2.2.2 Preparation and characterization of gold nanoparticle..............................................35
2.2.3 Designing of synthetic peptide......................36
2.2.4 Preparation of pH5N1-carrier conjugates.............36
2.2.5 Immunization of mice................................37
2.2.6 Enzyme-linked immunosorbent assay...................38
2.2.7 Inductively coupled plasma mass spectrometry........39
2.2.8 Statistical Analyses................................40
2.3 Result and Discussion.................................42
2.3.1 Preparation and analysis of pH5N1-GNP conjugates....42
2.3.2 The pH5N1-GNP conjugates induced focused and enhanced
antibody response.........................................44
2.3.3 The anti-pH5N1 antiserum recognized whole pH5N1 as epitope...................................................48
2.3.4 Antibody response of pH5N1-GNP conjugates is associated with the length of peptide.....................49
2.3.5 Antibody response of pFMDV-GNP conjugates is associatednwith the length of peptide.....................51
2.4 Conclusions...........................................55
References................................................56

Chapter 1
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Chapter 2
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[10] De Silva B S, Egodage K L and Wilson G S 1999 Bioconjug Chem 10 496-501
[11] Riemer A B, Klinger M, Wagner S, Bernhaus A, Mazzucchelli L, Pehamberger H, Scheiner O, Zielinski C C and Jensen-Jarolim E 2004 J Immunol 173 394-401
[12] Rubinchik E and Chow A W 2000 Vaccine 18 2312-20
[13] Maitta R W, Datta K, Lees A, Belouski S S and Pirofski L A 2004 Infect Immun 72 196-208
[14] Beenhouwer D O, May R J, Valadon P and Scharff M D 2002 J Immunol 169 6992-9
[15] Fan J, Liang X, Horton M S, Perry H C, Citron M P, Heidecker G J, Fu T M, Joyce J, Przysiecki C T, Keller P M, Garsky V M, Ionescu R, Rippeon Y, Shi L, Chastain M A,
Condra J H, Davies M E, Liao J, Emini E A and Shiver J W 2004 Vaccine 22 2993-3003
[16] Tompkins S M, Zhao Z S, Lo C Y, Misplon J A, Liu T, Ye Z, Hogan R J, Wu Z, Benton K A, Tumpey T M and Epstein S L 2007 Emerg Infect Dis 13 426-35
[17] Shieh J J, Liang C M, Chen C Y, Lee F, Jong M H, Lai S S and Liang S M 2001 Vaccine 19 4002-10
[18] Paciotti G F, Myer L, Weinreich D, Goia D, Pavel N, McLaughlin R E and Tamarkin L 2004 Drug Deliv 11 169-83
[19] Loo C, Lin A, Hirsch L, Lee M H, Barton J, Halas N, West J and Drezek R 2004 Technol Cancer Res Treat 3 33-40
[20] Pissuwan D, Valenzuela S M and Cortie M B 2006 Trends Biotechnol 24 62-7
[21] Tomii A and Masugi F 1991 Jpn J Med Sci Biol 44 75-80
[22] Dean H J, Fuller D and Osorio J E 2003 Comp Immunol Microbiol Infect Dis 26 373-88
[23] Dykman L A, Sumaroka M V, Staroverov S A, Zaitseva I S and Bogatyrev V A 2004 Izv Akad Nauk Ser Biol 86-91
[24] Vasilenko O A, Staroverov S A, Yermilov D N, Pristensky D V, Shchyogolev S Y and Dykman L A 2007 Immunopharmacol Immunotoxicol 29 563-8
[25] Chithrani B D, Ghazani A A and Chan W C 2006 Nano Lett 6 662-8
[26] Chithrani B D and Chan W C 2007 Nano Lett 7 1542-50
[27] Hauck T S, Ghazani A A and Chan W C W 2008 Small 4 153-9
[28] De Jong W H, Hagens W I, Krystek P, Burger M C, Sips A J and Geertsma R E 2008 Biomaterials 29 1912-9
[29] Yu-Shiun Chen Y-C H, Ian Liau ,and G. Steve Huang 2009 Nanoscale Res Lett
[30] Sonavane G, Tomoda K and Makino K 2008 Colloids and Surfaces B-Biointerfaces 66 274-80
[31] Brown K R, Walter D G and Natan M J 2000 Chemistry of Materials 12 306-13
[32] Liu F K, Ker C J, Chang Y C, Ko F H, Chu T C and Dai B T 2003 Japanese Journal of Applied Physics Part 1-Regular Papers Short Notes & Review Papers 42 4152-8
[33] Slot J W and Geuze H J 1985 Eur J Cell Biol 38 87-93
[34] Beekman N J, Schaaper W M, Turkstra J A and Meloen R H 1999 Vaccine 17 2043-50
[35] Gharavi A E, Pierangeli S S, Colden-Stanfield M, Liu X W, Espinola R G and Harris E N 1999 J Immunol 163 2922-7
[36] Bittle J L, Houghten R A, Alexander H, Shinnick T M, Sutcliffe J G, Lerner R A, Rowlands D J and Brown F 1982 Nature 298 30-3
[37] Strohmaier K, Franze R and Adam K H 1982 J Gen Virol 59 295-306