碩士 / 國立臺灣大學 / 生命科學系 / 104 / Cryptocaryon irritans is a ciliate protozoan parasite which infects no less than 95 species of marine fish. The marine fish cultured densely in cages is vulnerable to C. irritans infection, and it leads to tremendous fish death. The combination vaccine was composed of C. irritans immobilization antigen (iAg) as antigen and the C terminus of C. irritans heat shock protein 70C (Hsp70C) as adjuvant. With both intramuscular injection and oral intubation, the combination vaccine confers high protection against cryptocaryonosis in Epinephelus coioides (grouper) fingerlings. Nevertheless, 17%, white spots (trophonts) remained on immunized fish with the necrotic lesion around the spots. Involved in parasite invasion, parasite cysteine proteases were usually demonstrated as antigen in anti-parasite vaccine. In addition, parasite cysteine proteases were potent triggers of Th2 immune responses. In the present study, the effect induced by C. irritans cathepsin B cysteine protease (CiCPB) as co-antigen in the combination vaccine was analyzed.
Two cathepsin cysteine proteases were cloned and characterized as CiCPB and CiCPL, respectively. CiCPB, CiCPL and grouper cysteine proteases (EcCPB and EcCPL) belong to different clusters phylogenetically, although they share 58% and 39% similarity in sequences, respectively. Real-time PCR revealed that CiCPB gene expression was significantly higher than CiCPL in theront stage. It was suggested that CiCPB is important in theront invasion, rather than CiCPL. The open reading frame of CiCPB was computationally reviewed to replace the 10 stop codons with suitable amino acids, and its GC content was intensified. Plasmid pcDNA3.1 was inserted with a signal peptide of grouper IgT heavy chain and CiCPB. The transfected grouper fin cells (GF-1 cells) successfully translated the insert protein.
Plasmid with iAg, Hsp70C and CiCPB was separately encapsulated in chitosan nanoparticles and orally intubated to grouper fingerlings with 20 μg/g fish body weight. The growth rate of fish body weight and body length during 24 days post immunization showed no significant difference among each group. The fish stomach and intestine tissue sections during 24 days post immunization showed no tissue lesion and hemorrhage. Fish was then challenged with a 50% lethal dose (LD50) of live theronts at 25 days post immunization. The combination vaccine group and combination vaccine with CiCPB group showed 100% and 8 % relative percent survival (RPS), respectively. Trophont burden on unit fish surface showed no difference among each group. Fish survived from last challenge suffered another 91% lethal dose (LD91) challenge at 31 days post immunization. The combination vaccine group and combination vaccine with CiCPB group showed 80% and 0% RPS, respectively.
In conclusion, the effect induced by CiCPB addition almost broke the protection induced by the combination vaccine in grouper fingerlings.
| Identifer | oai:union.ndltd.org:TW/104NTU05525001 |
| Date | January 2015 |
| Creators | Chia-Jung Lin, 林佳蓉 |
| Contributors | Yen-Ling Song, 宋延齡 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | en_US |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 71 |
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