弧菌是造成每年水產養殖重大經濟損失的主要病原菌之一，因此在高經濟物種常使用疫苗來預防弧菌爆發。其中重組蛋白疫苗應用高純度有效抗原，能提供時效較長的保護力。在前人研究中，利用免疫蛋白質體學已篩選到坎氏弧菌中一個具高度免疫原性的60 kDa 熱休克蛋白（Vibrio campbellii Heat shock protein 60, VcHSP60）。過去文獻指出 HSP60 可作為疫苗抗原，為小鼠及鮭魚提供免疫保護。因此，本研究欲探討VcHSP60作為次單位疫苗之效能以及其高度免疫原性之分子機制。表現並純化 VcHSP60 重組蛋白製備成疫苗，施打於石斑魚後，證實可誘發高力價之血清抗體，並且提供魚隻免疫保護力。此外，施打VcHSP60誘發之血清抗體可交叉辨識其他海洋病原菌之HSP60蛋白，顯示其具有開發為廣效疫苗之潛力。本研究更結合蛋白質晶體學及小角度X光散射技術，解析VcHSP60之蛋白質結構資訊，並應用交聯耦合質譜分析法確認VcHSP60抗原辨識位點於結構上之分佈，結果找到五個抗原片段具有較高的抗體結合能力，可能為VcHSP60之專一性抗原辨識位。這些研究結果有助於未來進一步針對VcHSP60疫苗開發進行研究，以減少由魚類疾病造成的經濟損失。

Vibrio bacteria are one of the major pathogens causing significant economic losses in aquaculture each year, so vaccines are often used in high-economic species to prevent Vibrio outbreaks. The recombinant protein vaccine uses a high-purity effective antigen to provide long-lasting protection. In previous studies, a highly immunogenic protein, Vibrio campbellii Heat shock protein 60 (VcHSP60), has been identified by immunoproteomics. Some studies also revealed that HSP60 can be used as a vaccine antigen to provide immune protection for mice and salmon. Therefore, this study was to investigate the efficacy of VcHSP60 as a subunit vaccine and its molecular mechanism of high immunogenicity. We expressed and purified the VcHSP60 recombinant protein, which was used to prepare a vaccine and then applied to grouper fish. The results confirmed that VcHSP60 did induce high serum titers of specific antibodies and provided fish immune protection against Vibrio campbellii. In addition, the VcHSP60-induced serum antibodies can cross-identify the HSP60 proteins of other marine pathogens, indicating its potential to be developed as a broad-spectrum vaccine. In this study, protein crystallography and small-angle X-ray scattering were utilized to solve the protein structure of VcHSP60, and the distribution of VcHSP60 epitops was identified by cross-linking coupled mass spectrometry. Five fragments on VcHSP6 showed higher antibody-binding affinity, indicating that these fragments might be the specific antibody-antigen recognition sites. These results could be ultilized to design the VcHSP60 vaccine for reducing the economic losses caused by fish diseases.

林伯峰，點帶石班魚兩腫瘤壞死因子-α（TNF -α1、TNF-α2）蛋白功能性分析探討，國立成功大學生物科技研究所碩士論文，2012。

林煜晟，石斑養殖上強毒株坎氏弧菌免疫抗原分析與疫苗開發，國立成功大學生物科技研究所碩士論文，2009。

郭宛靜，細菌性注射疫苗誘發石斑魚免疫因子基因表現及對保護力之影響，國立成功大學生物科技研究所碩士論文，2016。

張晉榮，比較巴斯德桿菌與坎氏弧菌之共同免疫原對於石斑魚抵抗坎氏弧菌感染之交叉保護能力研究，國立成功大學生物科技研究所碩士論文，2011。

游敏，點帶石斑魚兩腫瘤壞死因子-α 之蛋白質立體結構分析，國立成功大學生物科技與產業科學系碩士論文，2019。

Adu-Bobie, J., Capecchi, B., Serruto, D., Rappuoli, R., and Mariagrazia, P. Two years into reverse vaccinology. Vaccine 21, 605-610, 2003.

Adams, P. D., Afonine, P. V., Bunkóczi, G., Chen, V. B., Davis, I. W., Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J., and Grosse-Kunstleve, R. W. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallographica Section D: Biological Crystallography 66, 213-221. 2010.

Almanzar, G., Öllinger, R., Leuenberger, J., Onestingel, E., Rantner, B., Zehm, S., Cardini, B., van der Zee, R., Grundtman, C., and Wick, G. J. Autoreactive HSP60 epitope-specific T-cells in early human atherosclerotic lesions. Journal of Autoimmunity 39, 441-450, 2012.

Anfinsen, C.B. Principles that govern the folding of protein chains. Science 181, 223-230, 1973.
Arias, C., Macian, M., Aznar, R., Garay, E., and Pujalte, M. J. Low incidence of Vibrio vulnificus among Vibrio isolates from sea water and shellfish of the western Mediterranean coast. Journal of Applied Microbiology 86, 125-134, 1999.

Austin, B., and Zhang, X. H. Vibrio harveyi: a significant pathogen of marine vertebrates and invertebrates. Letters in Applied Microbiology 43, 119-124, 2006

Bailey, M.J., Hughes, C., and Koronakis, V. Increased distal gene transcription by the elongation factor RfaH, a specialized homologue of NusG. Molecular Mcrobiology 22, 729-737, 1996.

Bale, H. D., and Schmidt, P. W. Small-angle X-ray-scattering investigation of submicroscopic porosity with fractal properties. Physical Review Letters 53, 596, 1984.

Benjamin, D. C., and Perdue, S. S. Site-directed mutagenesis in epitope mapping. Methods 9, 508-515, 1996.

Bonfadini, M. X3DPROT: A tool for distributed 3D protein structure visualization and manipulation. Journal of Applied Crystallography 24, 946-950, 1991.

Braig, K., Otwinowski, Z., Hegde, R., Boisvert, D.C., Joachimiak, A., Horwich, A.L., and Sigler, P.B. The crystal structure of the bacterial chaperonln GroEL at 2.8 Å. Nature 371, 578, 1994.

Cao, Y., Ohwatari, N., Matsumoto, T., Kosaka, M., Ohtsuru, A., and Yamashita, S. TGF-β1 mediates 70-kDa heat shock protein induction due to ultraviolet irradiation in human skin fibroblasts. Pflügers Archiv 438, 239-244, 1999.

Chatterjee, S., and Haldar, S. Vibrio related diseases in aquaculture and development of rapid and accurate identification methods. Journal of Marine Science: Research and Development S, 1, 2012.

Chen, L., Xu, Y., Wong, W., Thompson, J. K., Healer, J., Goddard-Borger, E. D., Lawrence, M. C., and Cowman, A. F. Structural basis for inhibition of erythrocyte invasion by antibodies to Plasmodium falciparum protein CyRPA. ELife Sciences Publications 6, 21347, 2017.

Chen, Z., Peng, B., Wang, S., and Peng, X. Rapid screening of highly efficient vaccine candidates by immunoproteomics. Proteomics 4, 3203-3213, 2004.

Chitradevi, S., Kaur, G., Singh, K., Sugadev, R., and Bansal, A. Recombinant heat shock protein 60 (Hsp60/GroEL) of Salmonella enterica serovar Typhi elicits cross-protection against multiple bacterial pathogens in mice. Vaccine 31, 2035-2041, 2013.

Chittleborough, R. G. Environmental factors affecting growth and survival of juvenile western rock lobsters Panulirus longipes (Milne-Edwards). Marine and Freshwater Research 26, 177-196, 1975.

Ciervo, A., Visca, P., Petrucca, A., Biasucci, L. M., Maseri, A., and Cassone, A. Antibodies to 60-kilodalton heat shock protein and outer membrane protein 2 of Chlamydia pneumoniae in patients with coronary heart disease. Clinical and Diagnostic Laboratory Immunology 9, 66-74, 2002.

Claramunt, R. M., & Wahl, D. H.. The effects of abiotic and biotic factors in determining larval fish growth rates: a comparison across species and reservoirs. Transactions of the American Fisheries Society 129, 835-851, 2000.

Clarke, B., Newton, S., Carroll, A., Francis, M., Appleyard, G., Syred, A., Highfield, P., Rowlands, D., and Brown, F. Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein. Nature 330, 381, 1987.

Cohen, I. R., and Young, D. B. Autoimmunity, microbial immunity and the immunological homunculus. Immunology Today 12, 105-110, 1991.

Cornilescu, G., Marquardt, J. L., Ottiger, M., and Bax, A. Validation of protein structure from anisotropic carbonyl chemical shifts in a dilute liquid crystalline phase. Journal of the American Chemical Society 120, 6836-6837, 1998.

Cropper, M., and Griffiths, C. The interaction of population growth and environmental quality. The American Economic Review 84, 250-254, 1994.

Cui, M., Zhang, Q., Yao, Z., Zhang, Z., Zhang, H., and Wang, Y. Immunoglobulin M gene expression analysis of orange-spotted grouper, Epinephelus coioides, following heat shock and Vibrio alginolyticus challenge. Fish and Shellfish Immunology 29, 1060-1065, 2010.

Davies, D. R., and Cohen, G. H. Interactions of protein antigens with antibodies. Proceedings of the National Academy of Sciences of the United States of America 93, 7-12, 1996.

Defoirdt, T., Sorgeloos, P., and Bossier, P. Alternatives to antibiotics for the control of bacterial disease in aquaculture. Current Opinion in Microbiology 14, 251-258, 2011.

DeMaria, A., Johns, M. A., Berberich, H., and McCabe, W. R. Immunization with rough mutants of Salmonella minnesota: initial studies in human subjects. Journal of Infectious Diseases 158, 301-311, 1988.

Diamantis, N., and Banerji, U. Antibody-drug conjugates-an emerging class of cancer treatment. British Journal of Cancer 114, 362, 2016.

Dyksterhuis, L. B., Baldock, C., Lammie, D., Wess, T. J., and Weiss, A. S. Domains 17-27 of tropoelastin contain key regions of contact for coacervation and contain an unusual turn-containing crosslinking domain. Matrix Biology 26, 125-135, 2007.

Edelman, R. Vaccine adjuvants. Reviews of Infectious Diseases 2, 370-383, 1980.

Edman, P. Method for determination of the amino acid sequence in peptides. Archives of Biochemistry and Biophysics 4, 283-293, 1950.
Elias, D., Meilin, A., Ablamunits, V., Birk, O. S., Carmi, P., Könen-Waisman, S., and Cohen, I. R. Hsp60 peptide therapy of NOD mouse diabetes induces a Th2 cytokine burst and downregulates autoimmunity to various β-cell antigens. Diabetes 46, 758-765, 1997.

Feltkamp, M. C., Smits, H. L., Vierboom, M. P., Minnaar, R. P., De Jongh, B. M., Drijfhout, J. W., Schegget, J. T., Melief, C. J., and Kast, W. M. Vaccination with cytotoxic T lymphocyte epitope‐containing peptide protects against a tumor induced by human papillomavirus type 16‐transformed cells. EuropeanJournal of Immunology 23, 2242-2249, 1993.

Feng, Y., Pan, X., Sun, W., Wang, C., Zhang, H., Li, X., Ma, Y., Shao, Z., Ge, J., and Zheng, F. J. Streptococcus suis enolase functions as a protective antigen displayed on the bacterial cell surface. The Journal of Infectious Diseases 200, 1583-1592, 2009.
Ferriola, P. C., Cody, V., and Middleton, E. Protein kinase C inhibition by plant flavonoids: kinetic mechanisms and structure-activity relationships. Biochemical Pharmacology 38, 1617-1624, 1989.

Franke, D., and Svergun, D. I. DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. Journal of Applied Crystallography 42, 342-346, 2009.

Frans, I., Michiels, C. W., Bossier, P., Willems, K., Lievens, B., and Rediers, H. Vibrio anguillarum as a fish pathogen: virulence factors, diagnosis and prevention. Journal of Fish Diseases 34, 643-661, 2011.

Frauenfelder, H., Petsko, G. A., and Tsernoglou, D. Temperature-dependent X-ray diffraction as a probe of protein structural dynamics. Nature 280, 558, 1979.

Feltkamp, M. C., Smits, H. L., Vierboom, M. P., Minnaar, R. P., De Jongh, B. M., Drijfhout, J. W., Schegget, J. T., Melief, C. J., and Kast, W. M. Vaccination with cytotoxic T lymphocyte epitope‐containing peptide protects against a tumor induced by human papillomavirus type 16‐transformed cells. European Journal of Immunology 23, 2242-2249, 1993.

Frisk, A., Ison, C., and Lagergård, T. GroEL heat shock protein of Haemophilus ducreyi: association with cell surface and capacity to bind to eukaryotic cells. Infection and Immunity 66, 1252-1257, 1998.

Garduño, R. A., Garduño, E., and Hoffman, P. S. Surface-associated hsp60 chaperonin of Legionella pneumophila mediates invasion in a HeLa cell model. Infection and Immunity 66, 4602-4610, 1998.

Germain, R. N., and Margulies, D. H. The biochemistry and cell biology of antigen processing and presentation. Annual Review of Immunology 11, 403-450, 1993.

Guet, J., Lin, Q., Linares-Solano, A., and de Lecea, C. Characterization of activated carbon: an approach to the activation process by SAXS and optical microscopy. Studies in Surface Science and Catalysis 66, 379-387. 1991.
Hennequin, C., Porcheray, F., Waligora-Dupriet, A. J., Collignon, A., Barc, M. C., Bourlioux, P., and Karjalainen, T. GroEL (Hsp60) of Clostridium difficile is involved in cell adherence. Microbiology 147, 87-96, 2001.

Hermeling, S., Crommelin, D. J., Schellekens, H., and Jiskoot, W. Structure-immunogenicity relationships of therapeutic proteins. Pharmaceutical Research 21, 897-903, 2004.

Ho, L. P., Lin, J. H. Y., Liu, H. C., Chen, H. E., Chen, T. Y., and Yang, H. L. Identification of antigens for the development of a subunit vaccine against Photobacterium damselae ssp. piscicida. Fish and Shellfish Immunology 30, 412-419, 2011.

Huang, J., and Honda, W. CED: a conformational epitope database. BioMed Central Immunology 7, 7, 2006.

Huang, J. B., Wu, Y. C., and Chi, S. C. Dietary supplementation of Pediococcus pentosaceus enhances innate immunity, physiological health and resistance to Vibrio anguillarum in orange-spotted grouper (Epinephelus coioides). Fish and Shellfish Immunology 39, 196-205, 2014.

Jones, S., and Thornton, J. M. Principles of protein-protein interactions. Proceedings of the National Academy of Sciences of the United States of America 93, 13-20, 1996.

Jyot, J., Gautam, J. K., Raje, M., and Ghosh, A. Localization of DnaK and GroEL in Vibrio cholerae. Federation of European Microbiological Societies Microbiology Letters 172, 165-171, 1999.

Kabsch, W., and Sander, C. Dictionary of protein secondary structure: pattern recognition of hydrogen‐bonded and geometrical features. Biopolymers: Original Research on Biomolecules 22, 2577-2637. 1983.

Kaleta, E. F., and Taday, E. M. Avian host range of Chlamydophila spp. based on isolation, antigen detection and serology. Avian Pathology 32, 435-462, 2003.

Karas, M., and Hillenkamp, F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Analytical Chemistry 60, 2299-2301, 1988.

Kavran, J. M., and Leahy, D. J. Coupling antibody to cyanogen bromide-activated sepharose. Methods in Enzymology 541, 27-34, 2014.

Kawai, K., Liu, Y., Ohnishi, K., and Oshima, S. I. A conserved 37 kDa outer membrane protein of Edwardsiella tarda is an effective vaccine candidate. Vaccine 22, 3411-3418, 2004.

Kendrew, J. C., Bodo, G., Dintzis, H. M., Parrish, R., Wyckoff, H., and Phillips, D. C. A three-dimensional model of the myoglobin molecule obtained by X-ray analysis. Nature 181, 662-666, 1958.

Kim, Y. B., Okuda, J., Matsumoto, C., Takahashi, N., Hashimoto, S., and Nishibuchi, M. Identification of Vibrio parahaemolyticus strains at the species level by PCR targeted to the toxR gene. Journal of Clinical Microbiology 37, 1173-1177, 1999.

Kloetzel, P. M., and Ossendorp, F. Proteasome and peptidase function in MHC-class-I-mediated antigen presentation. Current Opinion in Immunology 16, 76-81, 2004.

Koberstein, J. T., Morra, B., and Stein, R. S. The determination of diffuse-boundary thicknesses of polymers by small-angle X-ray scattering. Journal of Applied Crystallography 13, 34-45, 1980.

Konarev, P. V., Petoukhov, M. V., Volkov, V. V., and Svergun, D. I. ATSAS 2.1, a program package for small-angle scattering data analysis. Journal of Applied Crystallography 39, 277-286, 2006.

Lee, J. Y., Yi, N. N., Kim, U. S., Choi, J. S., Kim, S. J., and Choi, J. I. Porphyromonas gingivalis heat shock protein vaccine reduces the alveolar bone loss induced by multiple periodontopathogenic bacteria. Journal of Periodontal Research 41, 10-14, 2006.

Li, H., Xiong, X. P., Peng, B., Xu, C. X., Ye, M. Z., Yang, T. C., Wang, S. Y., and Peng, X. X. Identification of broad cross-protective immunogens using heterogeneous antiserum-based immunoproteomic approach. Journal of Proteome Research 8, 4342-4349, 2009.

Lichtenstein, L. M., and Osler, A. G. Studies on the mechanisms of hypersensitivity phenomena: IX. Histamine release from human leukocytes by ragweed pollen antigen. Journal of Experimental Medicine 120, 507-530, 1964.

Mariuzza, R. A., Phillips, S. E. V., and Poljak, R. J. The structural basis of antigen-antibody recognition. Annual Review of Biophysics and Biophysical Chemistry 16, 139-159, 1987.

Mattson, G., Conklin, E., Desai, S., Nielander, G., Savage, M. D., and Morgensen, S. A practical approach to crosslinking. Molecular Biology Reports 17, 167-183, 1993.

Matz, J. M., Blake, M. J., Tatelman, H. M., Lavoi, K. P., and Holbrook, N. J. Characterization and regulation of cold-induced heat shock protein expression in mouse brown adipose tissue. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 269, R38-R47, 1995.
McCullough, K. C. and Summerfield, A. Basic concepts of immune response and defense development. Institute for Laboratory Animal Research Journal 46, 230-240, 2005.

Michaëlsson, J., De Matos, C. T., Achour, A., Lanier, L. L., Kärre, K., and Söderström, K. A signal peptide derived from hsp60 binds HLA-E and interferes with CD94/NKG2A recognition. Journal of Experimental Medicine, 196, 1403-1414, 2002.

Mutharia, L. M., Klinck, J., Yamaguchi, H., and Davey, M. Purification, characterization and immunochemical properties of a novel 60-kDa protein of Vibrio anguillarum strains. Federation of European Microbiological Societies Microbiology Letters 168, 111-117, 1998.

Nagy, G., Emo, L., and Pál, T. Strategies for the development of vaccines conferring broad-spectrum protection. International Journal of Medical Microbiology 298, 379-395, 2008.

Nisemblat, S., Yaniv, O., Parnas, A., Frolow, F., and Azem, A. Crystal structure of the human mitochondrial chaperonin symmetrical football complex. Proceedings of the National Academy of Sciences of the United States of America 112, 6044-6049, 2015.

Novotny, J., and Sharp, K. Electrostatic fields in antibodies and antibody/antigen complexes. Progress in Biophysics and Molecular Biology 58, 203-224, 1992.

Novriadi, R. Vibriosis in aquaculture. Omni-Akuatika 1, 12, 2016.

Ostermann, J., Horwich, A. L., Neupert, W., and Hartl, F. U. Protein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis. Nature 341, 125, 1989.

Overholt, E. L., Tigertt, W., Kadull, P. J., Ward, M. K., David, C. N., Rene, R. M., Salzman, T. E., and Stephens, M. An analysis of forty-two cases of laboratory-acquired tularemia: treatment with broad spectrum antibiotics. The American Journal of Medicine 30, 785-806, 1961.
Parker, C. E., and Tomer, K. B. MALDI/MS-based epitope mapping of antigens bound to immobilized antibodies. Molecular Biotechnology 20, 49-62, 2002.

Pauza, C. D., Trivedi, P., Wallace, M., Ruckwardt, T. J., Le Buanec, H., Lu, W., Bizzini, B., Burny, A., Zagury, D., and Gallo, R. C. Vaccination with tat toxoid attenuates disease in simian/HIV-challenged macaques. Proceedings of the National Academy of Sciences of the United States of America 97, 3515-3519, 2000.

Peter, J. F., and Tomer, K. B. A general strategy for epitope mapping by direct MALDI-TOF mass spectrometry using secondary antibodies and cross-linking. Analytical Chemistry 73, 4012-4019, 2001.

Plant, K. P., LaPatra, S. E., and Cain, K. D. Vaccination of rainbow trout, Oncorhynchus mykiss (Walbaum), with recombinant and DNA vaccines produced to Flavobacterium psychrophilum heat shock proteins 60 and 70. Journal of Fish Diseases 32, 521-534, 2009.

Pujalte, M., Sitjà-Bobadilla, A., Macián, M., Belloch, C., Alvarez-Pellitero, P., Pérez-Sánchez, J., Uruburu, F., and Garay, E. Virulence and molecular typing of Vibrio harveyi strains isolated from cultured dentex, gilthead sea bream and European sea bass. Systematic and Applied Microbiology 26, 284-292, 2003.

Quintana, F. J., and Cohen, I. R. The HSP60 immune system network. Trends in Immunology 32, 89-95, 2011.

Ranford, J. C., Coates, A. R., and Henderson, B. Chaperonins are cell-signalling proteins: the unfolding biology of molecular chaperones. Expert Reviews in Molecular Medicine 2, 1-17, 2000.

Rappuoli, R. Reverse vaccinology, a genome-based approach to vaccine development. Vaccine 19, 2688-2691, 2001.

Rinaudo, C. D., Telford, J. L., Rappuoli, R., and Seib, K. L. Vaccinology in the genome era. The Journal of Clinical Investigation 119, 2515-2525, 2009.
Sakai, M. Current research status of fish immunostimulants. Aquaculture, 172, 63-92, 1999.

Sasaki, T., Hohenester, E., Göhring, W., and Timpl, R. Crystal structure and mapping by site‐directed mutagenesis of the collagen‐binding epitope of an activated form of BM‐40/SPARC/osteonectin. The European Molecular Biology Organization Journal 17, 1625-1634, 1998.

Sattentau, Q., Arthos, J., Deen, K., Hanna, N., Healey, D., Beverley, P., Sweet, R., and Truneh, A. Structural analysis of the human immunodeficiency virus-binding domain of CD4. Epitope mapping with site-directed mutants and anti-idiotypes. Journal of Experimental Medicine 170, 1319-1334, 1989.

Savan, R., Aman, A., Nakao, M., Watanuki, H., and Sakai, M. Discovery of a novel immunoglobulin heavy chain gene chimera from common carp (Cyprinus carpio L.). Immunogenetics 57, 458-463, 2005.

Schmidt, T., Bergner, A., and Schwede, T. Modelling three-dimensional protein structures for applications in drug design. Drug Discovery Today 19, 890-897, 2014.

Seib, K. L., Zhao, X., and Rappuoli, R. Developing vaccines in the era of genomics: a decade of reverse vaccinology. Clinical Microbiology and Infection 18, 109-1162012.

Sela, M., Schechter, B., Schechter, I., and Borek, F. Antibodies to sequential and conformational determinants. Cold Spring Harbor Symposia on Quantitative Biology 32, 537-545, 1967.

Sette, A., and Fikes, J. Epitope-based vaccines: an update on epitope identification, vaccine design and delivery. Current Opinion in Immunology 15, 461-470, 2003.

Shpigel, E., Elias, D., Cohen, I. R., and Shoseyov, O. Production and Purification of a Recombinant Human hsp60 Epitope Using the Cellulose-Binding Domain in Escherichia coli. Protein Expression and Purification 14, 185-191, 1998.
Siegrist, C. A. Vaccine immunology. Vaccines 5, 17-36, 2008.

Sivaram, V., Babu, M. M., Immanuel, G., Murugadass, S., Citarasu, T., and Marian, M. P.. Growth and immune response of juvenile greasy groupers (Epinephelus tauvina) fed with herbal antibacterial active principle supplemented diets against Vibrio harveyi infections. Aquaculture 237, 9-20, 2004.

Sommerset, I., Krossoy, B., Biering, E., and Frost, P. Vaccines for fish in aquaculture. Expert Review of Vaccines 4, 89-101, 2005.

Sorum, U., and Damsgård, B. Effects of anaesthetisation and vaccination on feed intake and growth in Atlantic salmon (Salmo salar L.). Aquaculture 232, 333-341, 2004

Spinello, A., Ortore, M., Spinozzi, F., Ricci, C., Barone, G., Gammazza, A. M., and Piccionello, A. P. Quaternary structures of GroEL and naïve-Hsp60 chaperonins in solution: A combined SAXS-MD study. Royal Society of Chemistry Advances 5, 49871-49879, 2015.

Stoddard, B. L., and Koshland, D. E. Prediction of the structure of a receptor–protein complex using a binary docking method. Nature 358, 774, 1992.

Svergun, D. I. and Koch, M. H. Advances in structure analysis using small-angle scattering in solution. Current Opinion in Structural Biology 12, 654-660, 2002.

Svergun, D. I. and Koch, M. H. Small-angle scattering studies of biological macromolecules in solution. Reports on Progress in Physics 66, 1735-1782, 2003.

Svergun, D. I., Petoukhov, M. V. and Koch, M. H. Determination of domain structure of proteins from X-ray solution scattering. Biophysical Journal 80, 2946-2953, 2001.

Tanton, T. W. Environmental factors affecting the yield of tea (Camellia sinensis). I. Effects of air temperature. Experimental Agriculture 18, 47-52, 1982.

Tong, J. C., Tan, T. W., and Ranganathan, S. Methods and protocols for prediction of immunogenic epitopes. Briefings in Bioinformatics 8, 96-108, 2006

Topf, M., Lasker, K., Webb, B., Wolfson, H., Chiu, W., and Sali, A. Protein structure fitting and refinement guided by cryo-EM density. Structure 16, 295-307, 2008.

Trewhella, J., Carlson, V. A., Curtis, E. H. and Heidorn, D. B. Differences in the solution structures of oxidized and reduced cytochrome c measured by small-angle X-ray scattering. Biochemistry 27, 1121-1125, 1988.

Vilasi, S., Carrotta, R., Mangione, M. R., Campanella, C., Librizzi, F., Randazzo, L., Martorana, V., Gammazza, A. M., Ortore, M. G., and Vilasi, A. Human Hsp60 with its mitochondrial import signal occurs in solution as heptamers and tetradecamers remarkably stable over a wide range of concentrations. Public Library of Science One 9, e97657, 2014.

Watts, C. Capture and processing of exogenous antigens for presentation on MHC molecules. Annual Review of Immunology 15, 821-850, 1997.

Wilfert, C. M.. Epidemiology of Haemophilus influenzae type b infections. Pediatrics 85, 631-635, 1990.

Wilhelm, V., Soza, C., Martínez, R., Rosemblatt, M., Burzio, L.O., and Valenzuela, P. D. Production and immune response of recombinant Hsp60 and Hsp70 from the salmon pathogen Piscirickettsia salmonis. Biological Research 38, 69-82, 2005.

Wunner, W. H., Dietzschold, B., Curtis, P. J., and Wiktor, T. J. Rabies subunit vaccines. Journal of General Virology 64, 1649-1656, 1983.

Yeh, Y. Q., Liao, K. F., Shih, O., Shiu, Y. J., Wu, W. R., Su, C. J., Lin, P. C. and Jeng, U. S. Probing the acid-induced packing structure changes of the molten globule domains of a protein near equilibrium unfolding. The Journal of Physical Chemistry Letters 82, 470-477, 2017.