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博碩士論文 etd-0529115-094154 詳細資訊
Title page for etd-0529115-094154
論文名稱
Title
龍膽石斑神經壞死病毒似病毒顆粒晶體穩定度之研究
The Studies on Crystal Stability of Virus-like Particle from Dragon Grouper Betanodavirus
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
183
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2015-06-09
繳交日期
Date of Submission
2015-06-29
關鍵字
Keywords
龍膽石斑神經壞死病毒、交聯、結晶、似病毒顆粒
virus-like particles, Dragon grouper nervous necrosis virus, crystallization, cross-linking
統計
Statistics
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中文摘要
龍膽石斑神經壞死病毒 (Dragon grouper nervous necrosis virus; DGNNV) 在病毒的分類上屬於 Nodaviridae 科 betanodavirus 屬,會對龍膽石斑魚 (dragon grouper; Epinephelus lanceolatus) 造成病毒性神經壞死病 (viral Nervous Necrosis;VNN),在魚仔期跟幼魚期死亡率高達100%。 在本篇的研究以大腸桿菌 (E. coli) 來大量表現龍膽石斑神經壞死病毒外殼蛋白的似病毒顆粒 (virus-like particles, VLPs),本研究以磷酸鹽緩衝溶液 (Phosphate Buffer) 純化似病毒顆粒,交聯劑Disuccinimidyl suberate (DSS) 作處理,可以使晶體更穩固,並可以得到更高解析度的繞射點。另外藉由 Poly-His tagged 選植株 VLPs 和胺基酸的突變株,發現 C338-C6H 不形成晶體,而點突變 N25R29A 的似病毒顆粒形成的晶體體積較大且完整。
Abstract
Dragon grouper nervous necrosis virus (DGNNV), a betanodavirus, is the cause of viral nervous necrosis in dragon grouper (Epinephelus lanceolatus). In larvae and juveniles, mortality is up amount 100%. The capsid protein of the DGNNV form virus-like particles (VLPs) that were expressed in Escherichia coli. So far, Crystals of VLPs diffracted X-rays to ~7.5 Å resolution (Luo and Lin, 2014). In this study, VLPs purified in phosphate buffer were cross-linked with DSS grown to crystals of higher resolution of diffracted X-rays. Among poly-His tagged VLPs and point-mutation, C338C6H can not form crystals while N25R29A mutant resulted in bigger size. VLPs for studies of DGNNV VLPs protein structure and function.
目次 Table of Contents
目錄
謝誌 I
摘要 II
Abstract III
目錄 IV
表目錄 VII
圖目錄 VIII
壹、前言 1
一、台灣石斑魚現今概況 1
二、魚類神經壞死病毒之感染 3
三、Betanodavirus 4
四、Betanodavirus 傳播 5
五、神經壞死病毒的感染途徑 7
六、Nodavirus 基因體及其遺傳物質之特性 9
七、魚類神經壞死病毒之防疫 16
八、似病毒顆粒 (Virus-like particles, VLPs) 的文獻 18
九、病毒結構生物學的發展 24
十、冷凍電子顯微鏡 26
十一、X-ray 蛋白質晶體學 27
十二、研究動機 32
貳、材料與方法 34
一、大腸桿菌表現 DGNNV VLPs 的外殼蛋白 34
二、DGNNV VLPs 的純化-蔗糖梯度離心法 35
三、連續式分光光度計分析 37
四、蛋白質定量 (Lowry method) 39
五、DGNNV VLPs 似病毒顆粒的拆裝與固定 40
六、DGNNV VLPs 蛋白質交聯處理 40
七、SDS-PAGE 蛋白質電泳 42
八、穿透式電子顯微鏡的觀察 43
九、VLP 的結晶 44
十、晶體穩定性的檢測 46
十一、晶體的交聯處理 48
十二、DGNNV VLPs 蛋白質固化條件的調整 48
十三、蛋白質晶體的繞射實驗 50
十四、預測龍膽石斑神經壞死病毒 (DGNNV) 之 3D 結構 51
十五、氨基酸序列結構的預測 52
十六、片段氨基酸結構的重組 54
十七、預測龍膽石斑神經壞死病毒外殼蛋白 3D 結構預測的微調 54
十八、病毒主殼層骨架的選擇 55
十九 、建立病毒二十面體 56
參、 結果 57
一、DGNNV VLPs 似病毒顆粒之純化 57
二、不同種類緩衝溶液之 DGNNV VLPs 純化 57
三、純化後 DGNNV VLPs 似病毒顆粒的拆裝與固定 59
四、純化後 DGNNV VLPs 似病毒顆粒的交聯劑處理 60
五、DGNNV VLPs 蛋白質交聯條件的調整 63
六、不同結晶條件之晶體比較 63
七、結晶條件之微調 64
八、蛋白質晶體的檢測 65
九、不同 VLP 濃度對結晶影響 67
十、添加金屬離子對 DGNNV VLPs 晶體形成的影響 67
十一、晶體的抗凍處理 68
十二、DGNNV VLPs 晶體固化處理 69
十三、Poly-His tagged 選植株的結晶 72
十四、Mutation 突變株的結晶 72
十五、DGNNV 蛋白質 3D 結構預測 73
十六、DGNNV 蛋白質 3D 結構預測的微調 74

肆、討論 76
伍、參考文獻 84
陸、圖表 89
陸、附錄 154
參考文獻 References
王俊雄。2010。外殼蛋白之 N 端與雙硫鍵影響石斑神經壞死病毒顆粒組裝及熱穩定性之研究。國立中山大學海洋生物科技暨資源系,博士論文。
呂明偉、劉旺達、林全信。(1998)。台灣石斑(Epinephelus sp.)與其他魚類神經壞死病毒(NNV)外殼蛋白基因之比較。台灣水產年會。
呂明偉。2003。龍膽石斑神經壞死病毒外殼蛋白與其似病毒顆粒之研究。國立中山大學海洋資源系,博士論文。
沈士新、鄭安倉、劉秉忠、林正輝、冉繁華。2014。石斑魚產業發展與產銷分析,石斑Handbook of Grouper。水產養殖生技 NO.38:1-7
柯德宏。1993。石斑魚類的繁養殖。海水魚繁殖大全。養魚世界雜誌社。
陳俊宏。2008。視角取向對T = 3 病毒顆粒三維重建之影響。國立中山大學海洋生物科技暨資源學系碩士論文。
陳俊榮。2006。同步輻射蛋白質結晶學的近期發展與應用研究。物理雙月刊。廿八卷四期
鄧火土。1985。水產養殖。豐年叢書。
羅昱淳。2014。龍膽石斑神經壞死病毒似病毒顆粒的結晶與 X-ray 繞射之研究。國立中山大學海洋資源所,博士論文。
Arimoto, M., Mori, K., Nakai, T., Muroga, K. & Furusawa, I. (1993).Pathogenicity of the causative agent of viral nervous necrosis disease in striped jack, Pseudocaranx dentex (Bloch & Schneider). J Fish Dis 16, 461–469.
Badia-Martinez, D., Oksanen, H. M., Stuart, D. I. & Abrescia,N. G. A. (2013). Subcell. Biochem. 68, 203–246.
Baker, T. S., Newcomb, W. W., Olson, N. H., Cowsert, L. M., Olson, C. & Brown, J. C. (1991). Structures of bovine and human papillomaviruses. Analysis by cryoelectron microscopy and three-dimensional image reconstruction. Biophysical Journal 60, 1445-1456.
Ball LA (1995) Requirements for the self-directed replication of flock house virus RNA. J Virol 69: 2722
Bernal, J. & Crowfoot, D. (1934). Nature (London), 133, 794-795.
Bose-Basu, B., DeRose, E. F., Kirby, T. W., Mueller, G. A., Beard, W. A., Wilson, S. H. & London, R. E. (2004). Dynamic Characterization of a DNA Repair Enzyme: NMR Studies of [methyl-13C]Methionine-Labeled DNA Polymerase‚Biochemistry 43, 8911–8922
Boyes-Watson, J., Davidson, E. & Perutz, M. F. (1947). Proc. R. Soc.London Ser. A, 191, 83-132.
Bragg, W. L. & Perutz, M. F. (1952). Acta Cryst. 5, 277-283.
Breuil G, Bonami JR, Pepin JF, Pichot Y (1991) Viral infection (picorna-like virus) associated with mass mortalities in hatchery-reared sea bass (Dicentrarchus labrax) larvae and juveniles. Aquaculture 97: 109-116
Caparros-Wanderley, W., Savage, N., Hill-Perkins, M., Layton, G., Weber, J. & Davies, D. H. (1999). Intratype sequence variation among clinical isolates of the human papillomavirus type 6 L1 ORF, clustering of mutations and identification of a frequent amino acid sequence variant. The Journal of General Virology 80, 1025-1033.
Chen, Y., Ghim, S. J., Jenson, A. B. & Schlegel, R. (1998). Mutant canine oral papillomavirus L1 capsid proteins which form virus- like particles but lack native conformational epitopes. The Journal of General Virology 79, 2137-2146.
Chi SC, Lo CF, Lin SC. (2001) Characterization of grouper nervous necrosis virus. J Fish Dis. 24: 3-13.
Comps, M., Pepin, J. F., and Bonami, J. R. (1994). Purification and characterization of two fish encephalitis viruses (FEV) infecting Lates calcarifer and Dicentrarchus labrax. Aquaculture 123, 1-10.
Comps, M., Pepin, J. F., and Bonami, J. R. (1994). Purification and characterizationof two fish encephalitis viruses (FEV) infecting Lates calcarifer and Dicentrarchus labrax. Aquaculture 123, 1-10.
Crawford, S. E., Labbe, M., Cohen, J., Burroughs, M. H., Zhou, Y. J. & Estes, M. K. (1994). Characterization of virus-like particlesproduced by the expression of rotavirus capsid proteins in insect cells. Journal of Virology 68, 5945-5952.
Dauter Z. (2002).Curr Opin Struct Biol. 12, 674.
Delos SE, Montross L, Moreland RB, Garcea RL. (1993). Expression of the polyomavirus VP2 and VP3 proteins in insect cells: coexpression with the major capsid protein VP1 alters VP2/VP3 subcellular localization. Virology 194: 393–398.
Dong, X. F., Natarajan, P., Tihova, M., Johnson, J. E. & Schneemann, A. (1998). Particle polymorphism caused by deletion of a peptide molecular switch in a quasi-equivalent virus. Journal of Virology 72, 6024-6033.
Estes, M. K. & Cohen, J. (1989). Rotavirus gene structure and function. Microbiological Reviews 53, 410-449.
Favre, M. (1975). Structural polypeptides of rabbit, bovine and human papillomaviruses. Journal of Virology Methods 15, 1239-1247.
Fisher AJ, McKinney BR, Schneemann A, Rueckert RR, Johnson JE. (1993) Crystallization of viruslike particles assembled from flock house virus coat protein expressed in a baculovirus system. J Virol. 67: 2950-2953.
Forstova J, Krauzewicz N, Wallace S, Street AJ, Dilworth SM, Beard S, Griffin BE. (1993). Cooperation of structural proteins during late events in the life cycle of polyomavirus. J Virol 67: 1405-1413.
French, T. J. & Roy, P. (1990). Synthesis of blue-tongue virus (BTV) core-like particles by a recombinant baculovirus expressing the two major structural core proteins of BTV. Journal of Virology 64, 1530-1536.
Fukuda Y, Nguyen HD, Furuhashi M, Nakai T. (1996). Mass mortality of cultured sevenband grouper, Epinephelus septemfasciatus, associated with viral nervous necrosis. Fish Pathol. 31: 165-170.
Grotmol, S., Nerland, A. H., Biering, E., Totland, G. K. & Nishizawa, T. (2000). Characterization of the capsid protein gene from a nodavirus strain affecting the Atlantic halibut Hippoglossus hippoglossus and design of an optimal reverse-transcriptase polymerase chain reaction (RT-PCR) detection assay. Diseases of Aquatic Organisms 39, 79-88.
Grotmol, S., Totland, G.K., and Kryvi, H. (1997). Detection of a nodavirus-like agent in heart tissue from reared Atlantic salmon Salmo salar suffering from cardiac myopathy syndrome (CMS). Dis. Aquat. Org. 29, 79-84.
Guo, Y. X., Wei, T., Dallmann, K. & Kwang, J. (2003). Induction of caspase-dependent apoptosis by betanodavirus GGNNV and demonstration of protein • as an apoptosis inducer. Virology 308, 74-82.
Haffar OK, Smithgall MD, Moran PA, Travis BM, Zarling JM, Hu S L (1991) HIV-specific humoral and cellular immunity in rabbits vaccinated with recombinant human immunodeficiency virus-like gag-env particles. Virology 183: 487-495
Hagensee, M. E., Yaegashi, N. & Galloway, D. A. (1993). Self-assembly of human papillomavirus type 1 capsids by expression of the L1 protein alone or by coexpression of the L1 and L2 capsid proteins. Journal of Virology 67, 315-322.
Hendrickson W. A. (2000). Synchrotron crystallography. Trends in Biochemical Sciences. 12, 637
Laurent S Vautherot JF, Madelaine MF, Le Gall G, Rasschaert D (1994) Recombinant rabbit hemorrhagic disease virus capsid protein expressed in baculovirus self-assembles into viruslike particles and induces protection. J Virol 68: 6794-6798
Li, H., Li, W. X. & Ding, S. W. (2002). Induction and suppression of RNA silencing by an animal virus. Science 296, 1319-1321.
Li, M., Cripe, T. P., Estes, P. A., Lyon, M. K., Rose, R. C., and Garcea, R. L. (1997).Expression of the human papillomavirus type 11 capsid protein in Escherichia coli:characterization of protein domains involved in DNA binding and capsid assembly. J. Virol. 71, 2988–2995.
Lin, C. S., Lu, M. W., Tang, L., Liu, W., Chao, C. B., Lin, C. J.,Krishna, N. K., Johnson, J. E. & Schneemann, A. (2001).Characterization of virus-like particles assembled in a recombinant baculovirus system expressing the capsid protein of a fish nodavirus. Virology 290, 50-58.
Lin, C.S., Lu, M.W., Tang, L., Liu, W., Chao, C.B., Lin, C.J.,Krishna, N.K., Johnson, J.E., and Schneemann, A. (2001).Characterization of virus-like particles assembled in a recombinantbaculovirus system expressing the capsid protein of a fish nodavirus. Virology 290 (1), 50-8.
Lu, M.W. & Lin, C.S. (2003). Involvement of the terminus of grouper betanodavirus capsid protein in virus-like particle assembly. Archives of Virology 148, 345-355.
Luo YC, Wang CH, Wu YM, Liu W, Lu MW, *Lin CS. (2014). Crystallization andX-ray diffraction of virus-like particles from a piscine betanodavirus. Acta Crystallogr F Struct Biol Commun. 70:1080-6.
Marshall D, Schneemann A (2001) Specific packaging of nodaviral RNA2 requires the N-terminus of the capsid protein. Virology 285: 165-175
Mori, K., Nakai, Muroga, T. K., Arimoto, M., Mushiake, K., and Furusawa, I. (1992).Properties of new virus belonging to Nodaviridae found in larval striped jack (Pseudocaranx dentex) with nervous necrosis. Virology 187, 368-371.
Munday, B. L., Kwang, J. & Moody, N. (2002). Betanodavirus infection of teleost fish: a review. Journal of Fish Disease 25, 127–142.
Munday, B. L., Langdon, J. S., Hyatt, A. & Humphrey, J. D. (1992). Mass mortality associated with a viral-induced vacuolating encephalopathy and retinopathy of larval and juvenile barramundi, Lates calcarifer Bloch. Aquaculture 103, 197-211.
Mushiake, K., Nishizawa, T., Nakai, T., Furusawa I. & Muroga, K. (1994).Control of VNN in striped jack: selection of spwnerss based on the detection of SJNNV gene by polymerase chain reaction (PCR). Fish Pathology 29, 177-182.
Nagai, T. & Nishizawa, T. (1999). Sequence of the non-structural protein gene encoded by RNA1 of striped jack nervous necrosis virus. Journal of General Virology 80, 3019-3022.
Nardelli-Haefliger, D., Roden, R. B., Benyacoub, J., Sahli, R., Kraehenbuhl, J. P.,Schiller, J. T., Lachat, P., Potts, A., and De Grandi, P. (1997). Human papillomavirus type 16 virus-like particles expressed in attenuated Salmonella typhimurium elicit mucosal and systemic neutralizing antibodies in mice. Infect. Immun. 65, 3328–3336.
Nathan D. Burrows and R. Lee Penn. (2014). Towards an integrative structural biology approach: combining Cryo-TEM, X-ray crystallography, and NMR. J Struct Funct Genomics 15:117–124
Nguyen HD, Nakai T, Muroga K. (1996). Progression of striped jack nervous necrosis virus (SJNNV) infection in naturally and experimentally infected striped jack Pseudocaranx dentex larvae. Dis Aquat Org. 24: 99-105.
Nishizawa T, Furuhashi M, Nagai T, Nakai T, Muroga K. (1997).Genomic classfication of fish nodaviruses by molecular phylogenetic analysis of the coat protein gene. Appl Environ Microbiol. 63: 1633-1636.
Nishizawa, T., Furuhashi, M., Nagai, T., Nakai, T., and Muroga. K. (1997). Genomic classfication of fish nodaviruses by molecular phylogenetic analysis of the coat protein gene. Appl. Environ. Microbiol. 63,1633-1636.
Noad R, Roy P. (2003). Virus-like particles as immunogens. Trends Microbiol 11: 438-444.
Noad, R. & Roy, P. (2003). Virus-like particles as immunogens. TRENDS in Microbiology 11, 438-444.
Orlova EV, Saibil HR .(2011). Structural analysis ofmacromolecular assemblies by electron microscopy. Chem Rev 111:7710–7748
Parra F, Boga JA, Marin MS, Casais R (1993) The amino terminal sequence of VP60 from rabbit hemorrhagic disease virus supports its putative subgenomic origin. Virus Res 27: 219-228
Pfister, H., Gissmann, L. & Zur Hausen, H. (1977). Partial characterization of the proteins of human papilloma viruses (HPV) 1–3. Virology 83, 131-137.
Prasad, B. V., Wang, G. J., Clerx, J. P. & Chiu, W. (1988). Three-dimensional structure of rotavirus. Journal of Molecular Biology 199, 269-275.
Salunke DM, Caspar DL, Garcea RL. (1986). Self-assembly of purified polyomavirus capsid protein VP1. Cell 46: 895-904.
Schneemann A, Dasgupta R, Johnson JE, Rueckert RR. (1993) Use of recombinant baculoviruses in synthesis of morphologically distinct viruslike particles of flock house virus, a nodavirus. J Virol. 67: 2756-2763.
Schneemann A, Marshall D. (1998) Specific encapsidation of nodavirus RNAs is mediated through the C terminus of capsid precursor protein alpha. J Virol. 72: 8738-8746.
Skliris GP, Krondiris JV, Sideris DC, Shinn AP, Starkey WG,Richards RH. (2001). Phylogenetic and antigenic characterizationof new fish nodavirus isolates from Europe and Asia. Virus Res. 75:59-67.
Skliris, G. P., Krondiris, J. V., Sideris, D. C., Shinn, A. P., Starkey, W. G., and Richards, R. H. (2001). Phylogenetic and antigenic characterization of new fish nodavirus isolates from Europe and Asia. Virus Res. 75, 59-67.
Sommerset I, & Nerland A. (2004).Complete sequence of RNA1 and subgenomic RNA3 of Atlantic halibut nodavirus (AHNV). Diseases of Aquatic Organism 58, 117-125.
Stuart DI, Abrescia NG. (2013). From lows to highs: using low-resolution models to phase X-ray data. Acta Crystallogr D Biol Crystallogr. D 69, 2257–2265.
Suzich, J. A., Ghim, S., Palmer-Hill, F. J., White, W. I., Tamura, J. K., Bell, J. A.,Newsome, J. A., Bennet Jenson, A., and Schlegel, R. (1995). Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas. Proc. Natl. Acad. Sci. USA 92, 11553–11557.
Svergun, D. I., Richard, S., Koch, M. H., Sayers, Z., Kuprin, S. & Zaccai, G. (1998). Proc. Natl Acad. Sci. 95(5), 2267-2272.
Tang, L., Lin, C. S., Krishna, N. K., Yeager, M., Schneemann, A. & Johnson, J. E. (2002). Virus-like particles of a fish nodavirus display a capsid subunit domain organization different from that of insect nodaviruses. Journal of Virology 76, 6370-6375.
Trus, B. L., Roden, R. B., Greenstone, H. L., Vrhel, M., Schiller, J. T. & Booy, F. P. (1997). Novel structural features of bovine papillomavirus capsid revealed by a three-dimensional reconstruction to 9 Å resolution. Nature Structural Biology 4, 413-420.
Wagner R, Deml L, Fliessbach H, Wanner G, Wolf H. (1994). Assembly and extracellular release of chimeric HIV-1 Pr55gag retrovirus-like particles. Virology 200: 162-175.
Wizemann H, Brunn AV (1999) Purification of E. coli-expressed His-tagged hepatitis B core antigen by Ni2+ chelate affinity chromatography. J Virol Methods 77: 189-197
Won, Y.-Y., Davis, H.T., and Bates, F.S., Giant Wormlike Rubber.(1999).Micelles. Science. 283 (5404): p. 960-963.
Yoshikoshi, K. & Inoue, K. (1990). Viral nervous necrosis in hatchery-reared larval and juveniles of Japanese parrotfish, Oplegnathus fasciatus (Temminck & Schlegel). J Fish Dis 13, 69-77.
Zeltins, A. (2013). Construction and characterization of virus-like particles:a review.
Mol. Biotechnol. 53, 92-107.
Zhang, L. F., Zhou, J., Chen, S., Cai, L. L., Bao, Q. Y., Zheng, F. Y., Lu, J. Q., Padmanabha, J., Hengst, K., Malcolm, K. & Frazer, I. H. (2000). HPV6b virus like particles are potent immunogens without adjuvant in man. Vaccine 18, 1051-1058
Zhou, J., Stenzel, D. J., Sun, X. Y. & Frazer, I. H. (1993). Synthesis and assembly of infectious bovine papillomavirus particles in vitro. Journal of General Virology 74, 763-768.
Zimei Bu, Ralf Biehl, Michael Monkenbusch, Dieter Richter, and David J. E. Callaway. (2005). Coupled protein domain motion in Taq polymerase revealed by neutron spin-echo spectroscopy. Proc Natl Acad Sci U S A. 102 (49): 17646-17651.
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