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研究生:鍾玉東
研究生(外文):Yu-Tung Chung
論文名稱:新城病毒重組蛋白質疫苗與蛋白質佐劑之生產與研究
論文名稱(外文):Researches on the Development of Subunit Vaccines Against Newcastle Disease Virus And the Production of Proteinaceous Adjuvant Hsp72
指導教授:李靜宜 博士莊景凱 博士
指導教授(外文):Ching-Yi Lee,Ph.D.Chin-Kai Chuang,Ph.D.
口試委員:李文權 博士莊景凱 博士李靜宜 博士
口試委員(外文):Wen-Chuan Lee,Ph.D.Chin-Kai Chuang,Ph.D.Ching-Yi Lee,Ph.D.
口試日期:2013-05-31
學位類別:碩士
校院名稱:明新科技大學
系所名稱:化學工程與材料科技系碩士班
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:51
中文關鍵詞:新城病毒PTD-J-X融合蛋白次單位疫苗
外文關鍵詞:Newcastle disease virussubunit vaccine
相關次數:
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新城病毒 (Newcastle disease virus, NDV)為副黏液病毒科副黏液病毒屬之RNA封膜病毒。藉封膜上的融合蛋白 ( fusion,F) 和紅血球凝集素-神經胺酸酶蛋白( Hemagglutin-Neuroaminidase, HN) 兩個病毒醣蛋白與宿主細胞產生結合與細胞膜融合,促使病毒顆粒進入細胞內。為找出新城病毒封膜蛋白 HN較佳抗原片段,選殖四個 DNA片段到表現載體 pET22b-PTD1J1(Tb),轉形至 E. coli Rosetta-gami B(DE3) pLysSRARE。分別純化出 PTD-J-HN161-290、PTD-J-HN292-466、PTD-J-HN468-571和PTD-J-HN401-454/468-530蛋白質片段。對 PTD-J-HN融合蛋白質之 J 區特異結合之Hsp72 (誘發型Hsp70 ) 增強 PTD-J-X融合蛋白之抗原呈現性,將已選殖之小鼠熱休克蛋白72 (Heat shock protein 72, Hsp72),透過 5L發酵槽系統將培養製程放大並純化。將四種蛋白質片段添加 Hsp72後各免疫三隻小鼠,所得到的抗血清分別進行西方墨點法、免疫螢光染色及血球凝集試驗。西方墨點法結果顯示施打PTD-J- HN161-290、PTD-J-HN468-571 和PTD-J-HN401-454/468-530 所得到的抗血清皆可辨識 HeLa細胞所表現之全長 HN蛋白質;免疫螢光結果顯示僅PTD-J- HN468-571抗血清得到強且專一的螢光訊號;至於血球凝集試驗結果為:單一蛋白質片段之抗血清以 PTD-J - HN292-466中和抗體力價達32倍較佳;混合四種蛋白質片段之抗血清,其中和抗體力價可達256倍。綜合上述結果顯示:每個蛋白質片段對小鼠產生的免疫效果不同,期待未來可以利用不同片段的組合,以提升新城病毒重組蛋白質疫苗的保護效力。
(關鍵詞:新城病毒、PTD-J-X融合蛋白、次單位疫苗)
Newcastle disease virus (NDV) is an enveloped RNA virus of Paramyxoviridae family. Two envelope glycoproteins, Hemagglutinin-Neuroaminidase (HN) and Fusion (F), play major roles in the fusion of envelope membrane and cellular membrane to deliver viron particle into host cells. The interactions between HN and host cell glycoprotein(s) act the first step of virus infection. HN is a type II membrane protein with a transmembrane domain at N-terminal end, a middle stalk structure and C-terminal Head domain which is composed of six blade subdomains. It is believed that the Head domain contains the binding sites of host cell glycoprotein receptor and these sites could be used as epitopes to elicit antisera with virus neutralizing ability. A patented PTD-J-X/Hsp72 antigen presenting system had been developed and applied herein. Polypeptide X fused with the J domain can be presented to immune system more effectively via the Hsp72 which can associate J domain specifically and stimulate innate immune response by Tole-like receptor 2 (TLR2). A 5L fermentation method was established to prepare recombinant Hsp72 protein which was used as a protein type adjuvant. Four cDNA fragments encoding NDV HN Head subdomains, i.e., blade 1-2 (161-290), blade 3-4 (292-466), blade 5-6 (468-571) and blade 4-5 (401-454/468-530), were subcloned into pET22b-PTD1J1(Tb) plasmid to construct the pET22b-PTD1J1-HN161-290, pET22b-PTD1J1-HN292-466, pET22b-PTD1J1-HN468-571 and pET22b-PTD1J1-HN401-454/468-530 expression vectors in order to express the PTD-J-HN292-466, PTD-J-HN468-571, PTD-J-HN161-290, and PTD-J-HN401-454/468-530 recombinant proteins, respectively. Antisera elicited with the four PTD-J-HN/Hsp72 protein complexes could recognize full length HN overexpressed in HeLa cells demonstrated by western blotting and immunofluorescence methods. The highest neutralizing valency of the four antisera was 32, however, the mixture of them was 256 indicating that the Head domain contains multi-receptor binding sites.
中文摘要 v
英文摘要 vi
誌謝 vii
目錄 viii
圖目錄 xi
第一章 前言 1
第二章 文獻探討
2.1新城病毒 3
2.2疫苗之演進 4
2.3蛋白質佐劑Hsp72 5
2.4新城病毒重組蛋白質疫苗 6
第三章 材料與方法 7
3.1 實驗材料
3.1.1 化學藥品及抗體 7
3.1.2 載體 ( vector ) 8
3.1.3 勝任細胞 ( competent cell ) 8
3.1.4 商業套組 ( Kit ) 8
3.1.5 培養基及免染相關試劑配製 8
3.1.6 蛋白質生產及純化相關試劑 9
3.1.7 電泳相關緩衝溶液及配製 9
3.2實驗方法
3.2.1 cDNA 合成 11
3.2.2 新城病毒 HN重組蛋白質之表現 13
3.2.3 小鼠熱休克蛋白質表現及建置發酵槽量產平台 14
3.2.4 蛋白質純化與濃縮 15
3.2.5 蛋白質十二烷基硫酸鈉聚丙烯醯胺電泳 ( Sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE ) 16
3.2.6 西方墨點法 17
3.2.7 pCX-NDV HN-Flag2之建構與轉染 18
3.2.8 免疫試驗 18
3.2.9 免疫螢光染色法 18
3.2.10 血球凝集試驗
第四章 實驗結果
4.1 新城病毒HN重組蛋白質之純化及SDS-PAGE 21
4.2 小鼠熱休克蛋白Hsp72純化及SDS-PAGE 22
4.3 蛋白質定量 22
4.4 西方墨點法 23
4.5 免疫螢光染色法 23
4.6 血球凝集試驗
4.6.1 檢測病毒抗原力價- 1HAU及4HAU結果 24
4.6.2 血球凝集抑制試驗-單一抗血清結果 24
4.6.3血球凝集抑制試驗-混合不同片段之小鼠抗血清結果 24
第五章 討論 33
參考文獻 35
附錄 39


1.鍾玉東、林岱昀、蘇郁秀、莊景凱,”新城病病毒封膜蛋白HN之較加抗原性片段篩選”,中國畜牧學會會誌,41卷,2012,200。
2.林岱昀、蘇郁秀、鍾玉東、莊景凱,”熱休克蛋白70可增進融合在PTD-J domain上之NDV HN抗原基的免疫原性”,中國畜牧學會期刊,2012.12, 287-302。
3.王金和、王敏盈、李尚熾、李龍湖、潘銘正等,”生物技術科技教育改進計畫,第七章佐劑”,疫苗發展技術與實驗,動物基因轉殖與疫苗發展技術教學資源中心主編,教育部顧問室,1993,179-194。
4.翁振哲,新城雞瘟病毒的基質基因之選質、分析與快速檢測,國立嘉義大學農學院農業生物技術研究所,碩士論文,2005。
5. Aldous, E. W., J. K. Mynn, J. Banks, and D. J. Alexander. 2003. A molecular
epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus)
isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion
protein gene. Avian Pathol. 32: 239-256.
6. Alexander, D. J. 2011. Newcastle disease in the European Union 2000 to 2009. Avian Pathol. 40: 547-558.
7. Bousse, T. L., G. Taylor, S. Krishnamurthy, A. Portner, S. K. Samal, 1 and T. Takimodo. 2004. Biological significance of the second receptor binding site of Newcastle disease virus hemagglutinin-neuraminidase protein. J. Virol. 78: 13351-13355.
8. Cattoli, G., L. Susta, C. Terregino, and C. Brown. 2011. Newcastle disease: a review of field recognition and current methods of laboratory detection. J. Vet. Diagnostic Invest. 23: 637-656.
9. Corey, E. A., A. M. Nirza, E. Levendowsky, and R. M. Iorio. 2003. Fusion deficiency induced by mutations at the dimer interface in the Newcastle disease yirus hemagglutinin-neuraminidase is due to a temperature-dependent defect in
receptor binding. J. Viorol. 77: 6913-6922.
10. Connolly, S. A., G. P. Leser, T. S. Jardetzky, and R. A. Lamb. 2009. Bimolecular
complementation of paramyxovirus fusion and hemagglutinin-neuraminidase proteins enhances fusion: implications for the mechanism of fusion triggering. J. Virol. 83: 10857-10868.
11.Crennel, S., T. Takimodo, A. Portner, and G. Taylor. 2000. Crystal structure of the multifunctional paramyxovirus hemagglutinin-neuraminidase. Nature Structural Biol. 7: 1068-1074.
12. Gravel, K. A., L. W. McGinnes, J. Reitter, and T. G. Morrison. 2011. The transmembrane domain sequence affects the structure and function of the Newcastle disease virus fusion protein. J. Virol. 85: 3486-3497.
13. Hu, S., T. Wang, Y. Liu, C. Meng, X. Wang, Y. Wu, and X. Liu. 2010. Identification of a variable epitope on the Newcastle disease virus hemagglutinin-neuraminidase protein. Vet. Microbiol. 140: 92-97.
14. Iorio, R. M., G. M. Field, J. M. Sauvron, A. M. Mirza, R. Deng, P. J. Mahon, and J. P. Langedijk. 2001. Structural and functional relationship between the receptor recognition and neuraminidaseactivities of the Newcastle disease virus hemagglutinin-neuraminidase protein: receptor recognition 1 is dependent on
neuraminidase activity. J. Virol. 75: 1918-1927.
15. Iorio, R. M., R. J. Stddall, J. P. Sheehan, H. A. Bratt, R. L. Glickman, and A. M. Riel. 1991. Neutralization map of the hemagglutinin-neuaminidase glycoprotein of Newcastle disease virus: domain required by monoclonal antibodies that prevent receptor recognition. J. Virol. 65: 4999-5006.
16. Iorio, R. M., R. J. Syddall, R. L. Glickman, A. M. Riel, J. P. Sheehan, and M. A. Bratt. 1989. Identification of amino acid residues important to the Neuraminidase activity of the HN glycoprotein of Newcastle disease virus. Virol. 173: 196-204.
17. Kim, S. H., Y. Yan, and S. K. Samal. 2009. Role of the cytoplasmic tail amino acid sequences of Newcastle disease virus hemagglutinin-neuraminidase protein in viron incorporation, cell fusion, and pathogenicity. J. Virol. 83: 10250-10255.
18. Li, J., E. Quinlan, A. Mirza, and R. M. Iorio. 2004. Mutated form of the Newcastle disease virus hemagglutinin-neuraminidase interacts with the homologous fusion protein depite deficiencies in both receptor recognition and fusion promotion. J. Virol. 78: 5299-5310.
19. Mahon, P. J., A. M. Mirza, and R. M. Iorio. 2011. Role of the two sialic acid sites on the Newcastle disease virus HN protein in triggering the interaction with the fusion protein required for the promotion of fusion. J. Virol. 85: 12079-12082.
20. Mahon, P. J., A. M. Mirza, T. A. Musick, and R. M. Iorio. 2008. Engineered intermonomeric disulfide bonds in the globular domain of Newcastle disease virus hemagglutinin-neuraminidase protein: implications for the mechanism of fusion promotion. J. Virol. 82: 10386-10396.
21. McGinnes, L. W., and T. G. Morrison. 2006. Inhibition of receptor binding stabilizes Newcastle disease virus HN and F protein-containing complexes. J. Virol. 80: 2894-2903.
22. Miller, P. J., E. L. Decanini, and C. L. Afonso. 2010. Newcastle disease: evolution of genotypes and the related diagnostic challenges. Infection Genetics Evolution 10: 26-35.
23. Panda, A., S. Elankumaran, S. Krishnamurthy, Z. Huang, and S. K. Samal. 2004. Loss of N-linked glycoprotein from hemagglutinin-neuraminidase protein alters virulence. J. Virol. 78: 4965-4975.
24. Porotto, M., Z. Salah, I. DeVito, A. Talekar, S. G. Palmer, R. Xu, I. A. Wilson, and A. Moscona. 2012. The second receptor binding site of the clobular head of the Newcastle disease virus hemagglutinin-neuraminidase activates the stalk of multiple paramyxovirus receptor binding proteins to trigger fusion. J. Virol. 86: 5730-5741.
25. Yuan, P., K. A. Swanson, G. P. Leser, R. G. Paterson, R. A. Lamb, and T. S. Jardetzky. 2011. Structure of the Newcastle disease virus hemagglutinin- neuraminidase (HN) ectodomain reveals a four-helix bundle stalk. Proc. Natl. Acad. Sci. 108: 14920-14925.
26. Iorio RM, Glickman RL, Riel AM, Sheehan JP, Bratt MA.1989.Functional and neutralization profile of seven overlapping antigenic sites on the HN glycoprotein of Newcastle disease virus: monoclonal antibodies to some sites prevent viral attachment. Virus Res.13:245-61
27. Yang,C.Y.,H.K. Shieh,Y.L. Lin and P.C. Chang.1999. Newcastle disease virus isolated from recent outbreaks in Taiwan phylogenetically related to viruses ( genotype VII) from recent outbreaks in western Europe.Avian Dis.43:125-130.

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