표제지
목차
요약 5
SUMMARY 6
제1장 연구개발과제의 개요 9
제1절 연구개발의 목적 9
제2장 국내외 기술개발 현황 11
제3장 연구개발 수행내용 및 결과 14
제1절 다가백신 연구 14
1. 넙치 연쇄구균 특성 연구 14
2. 연쇄구균 다가백신 연구 19
제2절 서브유닛 백신 연구 24
1. 서브유닛 백신 신종 항원 발굴 24
2. 서브유닛 백신 임상효능 및 안전성 조사 24
제3절 주사백신용 adjuvant 연구 29
1. 주사백신용 adjuvant 효능 및 안전성 조사 29
제4절 경구백신 연구 35
1. 장점막 면역 연구 35
2. 리포좀 경구백신 개발 40
3. 바이러스 리포좀 경구백신 연구 61
4. 캡슐 경구백신 개발 63
5. 경구백신용 adjuvant 개발 70
제4장 목표달성도 및 관련분야에의 기여도 75
제1절 연구개발 목표의 달성도 75
제2절 관련분야 기여도 75
1. 국내 넙치 연쇄구균 발생 원인 구명 75
2. 국내 넙치 연쇄구균 유행 혈청형 다가백신 개발 및 민간 기술이전 75
3. 국내 최초 어류 경구백신 원천기술 개발 및 민간 기술이전 76
4. 나노 리포좀 경구백신 기술이전 및 산ㆍ연 공동연구를 통한 조기 산업화 추진 76
제5장 연구개발결과의 활용계획 77
제1절 기술적 측면 77
제2절 경제ㆍ산업적 측면 77
제3절 수산정책ㆍ사회적 측면 77
제6장 참고문헌 78
제7장 부록 80
판권기 83
Table 1. PCR primer and product size information in this study 15
Table 2. Phenotypic characteristics of S. parauberis isolated from olive flounder in this study 15
Table 3. Agglutination titers of representative serotype I strains belonged to three subserotypes against unabsorbed and absorbed antisera 16
Table 4. Serotyping of S. parauberis based on agglutination titers of FKCs against rabbit antisera 16
Table 5. Analysis of antibody response of serum from 4 different S. parauberis serotypes vaccinated fish 20
Table 6. Cumulative mortality of vaccinated fish were inoculated with 4 different S. parauberis strains 20
Table 7. Relative percent survival of vaccinated fish were inoculated with 4 different S. parauberis strains 21
Table 8. Experimental model for vaccine efficacy according to mixed with 3 different S. parauberis serotypes 22
Table 9. Experimental model for vaccine safety evaluation by antigen concentration for vaccination 22
Table 10. Cumulative mortality after challenge according to mixed with 3 different S. parauberis serotypes 22
Table 11. Relative percent survival after challenge according to mixed with 3 different S. parauberis serotypes 23
Table 12. Hematological characteristics after vaccination (mean±SD) 23
Table 13. Relative percent survival (RPS) of vaccinated olive flounder were inoculated with S. parauberis 26
Table 14. Hematological characteristics after injection with SimA protein adjuvant contained vaccine (mean±SD) 28
Table 15. Candidate of adjuvant for injectable vaccine 29
Table 16. Experimental model for 3 adjuvants of injectable vaccine 30
Table 17. Relative percent survival (RPS) of vaccinated olive flounder were inoculated with S. parauberis 32
Table 18. Comparison of relative survival rate after vaccination for 5 adjuvants of injectable vaccine 34
Table 19. Experimental model for antigen oral intubation and injection 38
Table 20. Relative percent of mortality in olive flounder inoculated with S. parauberis 39
Table 21. Experimental model for pilot scale manufacturing method of liposome oral vaccine 44
Table 22. Experimental model for clinical trial of liposome oral vaccine 47
Table 23. Experimental model for pilot scale manufacturing method of liposome oral vaccine 49
Table 24. Information of field efficacy testing farms for liposome oral vaccine prototypes 58
Table 25. Data for enconomic analysis of liposome oral vaccine 60
Table 26. Result of return on sales of oral vaccine experimental data 60
Table 27. Experimental model for liposome containing heat-killed VHSV oral vaccine 61
Table 28. Relative percent of survival of alginate and chitosan capsule vaccinated group of olive flounder 66
Table 29. Relative survival rate of olive flounder after the S. parauberis challenge post-oral administration 69
Table 30. Experimental model for chitosan and saponin adjuvants of oral vaccine 70
Table 31. Relative survival rate of chitosan and saponin vaccinated group of olive flounder 71
Table 32. Experimental model for GR01 and fucoidan adjuvants of oral vaccine 72
Table 33. Relative survival rate of GR01 (A) adjuvant vaccinated group of olive flounder 73
Table 34. Relative survival rate of fucoidan (B) adjuvant vaccinated group of olive flounder 73
Fig. 1. Serotyping method of S. parauberis in this study. A, slide agglutination test; B, microtiter test 14
Fig. 2. A neighbor-joining phylogenetic tree based on multiple alignments of 4 capsule polysaccharide genes, autolysin and M-like protein coding gene... 17
Fig. 3. Different inoculation sites of olive flounder. A, Intraperitoneal injection; B, Subcutaneous injection; C, Intravenous injection 18
Fig. 4. Survival rates of olive flounder inoculated with S. parauberis. A, Intraperitoneal injection; B, Subcutaneous injection; C, Intravenous injection 18
Fig. 5. Cumulative mortality of olive flounder Paralichthys olivaceus. inoculated with 7 S. parauberis strains 19
Fig. 6. Outline of S. parauberis vaccination for olive flounder in this study 20
Fig. 7. Cumulative mortality after injection with 4 S. parauberis (serotype Ia FPa4365, serotype Ib PR5, serotype Ic FB4870, serotype II FP2221/ injected... 20
Fig. 8. Analysis of antibody response of serum from 4 different serotypes S. parauberis FKC vaccinated fish performed by ELISA (post-vaccination 2 weeks) 22
Fig. 9. Histopathological investigation by vaccine administration (post-vaccination 1 week) 23
Fig. 10. Confirmation of expression in transformed E. coli BL21 (DE3) Codonplus-RIL cells was performed using purified recombinant FBP3 (A) and SimA... 24
Fig. 11. (A) Analysis of antibody response of serum from vaccinated fish performed by ELISA. (B) Survival rates of vaccinated olive flounder were inoculated... 24
Fig. 12. Analysis of antibody response of serum from FBP3 (A) and SimA (B) vaccinated fish performed by ELISA. (C) Survival rates of vaccinated olive flounder... 26
Fig. 13. qRT-PCR analysis of expression of immune-related genes. The head kidney were sampled at 1 and 7 days after vaccination. A, IL-8; B, MHC I-α;... 26
Fig. 14. Safety check after injection of S. parauberis SimA protein adjuvant (after 4 weeks). A, FKC only; B, FKC+SimA; C, FKC+aluminum hydroxide;... 27
Fig. 15. Survival rates of SimA protein adjuvant contained vaccine administered to olive flounder were inoculated with S. parauberis (A, C). Analysis of... 28
Fig. 16. Histopathological changes after injection with SimA protein adjuvant contained vaccine (post-vaccination 4 weeks) 29
Fig. 17. Specific antibody detection in fish serum by ELISA (Y value: OD 405). A, Fucoidan; B, Eungen and SOE-S 31
Fig. 18. Survival rates of vaccinated olive flounder were inoculated with S. parauberis. A, Fucoidan; B, Eungen and SOE-S 31
Fig. 19. qRT-PCR analysis of expression of immune-related genes. The head kidney were sampled at 1 and 3 weeks after vaccination (ANOVA, P〈0.05),... 32
Fig. 20. qRT-PCR analysis of expression of immune-related genes. The head kidney were sampled at 1 and 3 weeks after vaccination (ANOVA, P〈0.05),... 33
Fig. 21. Specific antibody detection in fish serum by ELISA. A; ELISA absorbance level (OD490nm) in vaccine treated group (FKC + adjuvant). B; ELISA... 34
Fig. 22. Comparison of histopathological changes after injection with 5 different adjuvants contained S. paraubers FKC. (A, cell infiltration; B, nuclear pyknosis;... 35
Fig. 23. Immune cells distribution after S. parauberis antigen oral intubation. (A) 1hr, (B) 1 week, (C) 2 week, (D) 3-4 weeks (green - CD83+, red - IgM+,... 36
Fig. 24. qRT-PCR analysis of expression of immune-related genes after S parauberis antigen intraperitoneal injection or oral intubation 37
Fig. 25. Analysis of antibody response of serum from antigen oral intubation serum (A) and intestinal mucus (B) performed by ELISA 38
Fig. 26. Analysis of antibody response of serum from antigen oral intubation. Serum (A) and mucus of skin (B) performed by ELISA 39
Fig. 27. (A) Outline of liposome coating method for S. parauberis FKC. (B) Liposome oral vaccine 40
Fig. 28. (A) Size and shape of liposome-coated S. parauberis FKC. (B) Antibody response after liposomes coated S. parauberis FKC were orally administered... 41
Fig. 29. (A) Elution graph (Paddle method). (B) Zeta potential of liposome 41
Fig. 30. Measurement of average particle size using dynamic light scattering. A, Probe-L; B, Probe-L-coating; C, Emulsion-L; D, Emulsion-L-coating 42
Fig. 31. Analysis of antibody response of serum from antigen oral intubation serum and intestinal mucus performed by ELISA 42
Fig. 32. Cumulative mortality (A) and relative survival rate (B) of olive flounder after the S. parauberis challenge post-oral administration 43
Fig. 33. (A) Preparation of liposome vaccine using peristaltic pump. (B) Elution test 43
Fig. 34. (A) Stability at temperature. (B) Elution graph in stimulated gastric fluid (Paddle method) 44
Fig. 35. Average size of liposome by mean of emulsion method. A, Emulsion; B, Emulsion-coating 44
Fig. 36. Analysis of antibody response of serum from liposome containing S. parauberis FKC vaccinated fish performed by ELISA. A, serum after 2 weeks;... 45
Fig. 37. Cumulative mortality (A) and relative survival rate (B) of olive flounder after the S. parauberis challenge post-oral administration 45
Fig. 38. Schematic diagram of the preparation of liposome oral vaccine 46
Fig. 39. Liposome formation according to methods and materials. A, lecithin-like emulsifier; B, soybean lecithin 46
Fig. 40. Measurement of size distribution using dynamic light scattering. A, soybean lecithin 4,000 rpm and thin-like emulsifier 2,000 rpm; B, ecithin-like... 47
Fig. 41. Analysis of antibody response of serum from liposome containing S. parauberis FKC vaccinated fish performed by ELISA. A, serum after 2 weeks;... 47
Fig. 42. Stability of liposome vaccine during the storage period (0-50 days). A, soybean lecithin 25℃; B, lecithin-like emulsifier 25℃; C, soybean lecithin... 48
Fig. 43. (A) Stability of liposome in simulated gastic fluid. (B) Stability against heat (25~80℃) 48
Fig. 44. Analysis of antibody response of serum from liposome containing S. parauberis FKC vaccinated fish performed by ELISA 50
Fig. 45. (A) Manufacturing equipment liposome oral vaccine. (B) liposome oral vaccine prototype 50
Fig. 46. (A) Stability against heat (25~80℃). (B) Measurement of size distribution of liposome oral vaccine prototype 51
Fig. 47. Stability of liposome oral vaccine prototype in sea water (A) and distilled water (B) 52
Fig. 48. (A) Analysis of antibody response of serum. (B) cumulative mortality of olive flounder after the S. parauberis challenge post-oral administration 52
Fig. 49. Antigen feeding dosage and experimental group for liposome vaccine 53
Fig. 50. Antigen feeding dosage and experimental group for liposome vaccine 53
Fig. 51. Analysis of antibody response of serum from liposome containing S. parauberis FKC vaccinated fish performed by ELISA. A, 1 week; B, 2 weeks;... 54
Fig. 52. Analysis of antibody response of intestine from liposome containing S. parauberis FKC vaccinated fish performed by ELISA. A, 1 week; B, 2 weeks;... 54
Fig. 53. qRT-PCR analysis of expression of IgM gene after S parauberisfrom liposome containing S. parauberis FKC vaccinated fish. A, 1 week; B, 2 weeks;... 55
Fig. 54. (A) Cumulative mortality of olive flounder after the S. parauberis challenge. (B) Hematological characteristics after vaccination 55
Fig. 55. Analysis of antibody response of serum from liposome containing S. parauberis FKC vaccinated fry fish performed by ELISA. A, 1 week; B, 2 weeks;... 56
Fig. 56. Analysis of antibody response of intestine from liposome containing S. parauberis FKC vaccinated fry fish performed by ELISA. A, 1 week; B, 2 weeks;... 56
Fig. 57. qRT-PCR analysis of expression of IgM gene after S parauberisfrom liposome containing S. parauberis FKC vaccinated fry fish. A, 1 week; B, 2 weeks;... 57
Fig. 58. (A) Cumulative mortality of olive flounder fry after the S. parauberis challenge. (B) Hematological characteristics after vaccination 57
Fig. 59. (A) Clinical trial plan for liposome oral vaccine. Three experimental farms in Jeju (B) and Pohang (C) 58
Fig. 60. Analysis of antibody response of serum from liposome oral vaccine prototypes vaccinated fish performed by ELISA. A, serum after 4 weeks;... 58
Fig. 61. (A) Survival rate of olive flounder after the S. parauberis challenge post-oral administration in 3 fish farms (for 2 weeks). (B) Cumulative mortality... 59
Fig. 62. Survival rate (A) and total biomass (B) of olive flounder after oral vaccine administration after 3 months in 3 fish farms 59
Fig. 63. (A) VHSV CPE, (B) VHSV titer tested by real-time PCR, (C) liposomes containing VHSV 61
Fig. 64. (A) Measurement of average size, (B) Zeta potential of liposome, (C) Zeta potential of chitosan coating liposome 62
Fig. 65. (A) Stability of liposome in simulated gastic fluid. (B) Stability against heat (25~80℃) 62
Fig. 66. Analysis of antibody response in serum (A) and intestinal mucus (B) performed by ELISA 62
Fig. 67. qRT-PCR analysis of expression of IgM gene. A, head-kidney; B, spleen; C, intestine 63
Fig. 68. Survival rate (A) and relative survival rate (B) of olive flounder after the VHSV challenge post-oral administration 63
Fig. 69. Size and shape of alginate-coated microparticle (A) and chitosan-coated nanoparticle (B) incorporating S. paraueris FKCs (200x) 64
Fig. 70. Relative antigen profiles for alginate and chitosan coated particles incoporating S. parauberis FKCs under artifical conditions. A, at pH2, 4, 6 and 8;... 64
Fig. 71. Relative antigen release profiles for alginate and chitosan coated particles incoporating S. parauberis FKCs under artifical conditions. A, alginate... 65
Fig. 72. Self-ingestion and digestion of commercial feed with alginate and chitosan coated particles incoporating S. parauberis FKCs in olive flounder.... 65
Fig. 73. Analysis of antibody response of serum from alginate and chitosan capsule (A) vaccinated fish performed by ELISA. (B) Survival rates of vaccinated... 66
Fig. 74. Relative antigen release profiles for alginate coated particles incoporating S. parauberis FKCs under artificial conditions. A, supplemented with... 67
Fig. 75. Relative antigen release profiles for alginate coated particles incoporating S. parauberis FKCs under artificial conditions. A, supplemented with... 68
Fig. 76. Size and shape of alginate coated particles incoporating S. parauberis FKCs. A, immediately after production; B, 3 month after production;... 68
Fig. 77. Analysis of antibody response of serum (A) and intestine (B) from alginate capsule vaccinated fish performed by ELISA 69
Fig. 78. Survival rates of olive flounder after the S. parauberis challenge post-oral administration (3 weeks). A, 1st experiment; B, 2nd experiment 69
Fig. 79. Analysis of antibody response after vaccination performed by ELISA. A, Post-vaccination 1 week; B, Post-vaccination 3 weeks 70
Fig. 80. Cumulative mortality of chitosan and saponin vaccinated group of olive flounder 71
Fig. 81. Analysis of antibody response after vaccination performed by ELISA. A, adjuvant GR01 (serum); B, adjuvant GR01 (intestine); C, adjuvant fucoidan... 72
Fig. 82. Survival rates of olive flounder after the S. parauberis challenge post-oral administration. A, adjuvant A, GR01; B, adjuvant B fucoidan 73
Fig. 83. qRT-PCR analysis of expression of immune-related genes after injection with GR01 or fucoidan adjuvants mixed S. parauberis FKC. A, Interleukin-6... 73
Fig. 84. Histopathological changes after injection with GR01 or fucoidan adjuvants mixed S. parauberis FKC (post-vaccination 4 weeks) 74