| Summary: | 碩士 === 國立成功大學 === 生物科技與產業科學系 === 107 === 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.
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