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Efficiency Evaluation of 5BT, a Genetically Recombinant Multi-epitope-protein, for Its Application as a Subunit Vaccine against Foot and Mouth Disease : 유전자 재조합형 다중 항원결정기 단백질, 5BT의 구제역 대응 아단위 백신으로써의 활용을 위한 효율성 평가

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dc.contributor.advisor강상기-
dc.contributor.authorHu Lina-
dc.date.accessioned2020-05-07T03:58:37Z-
dc.date.available2020-05-07T03:58:37Z-
dc.date.issued2020-
dc.identifier.other000000158710-
dc.identifier.urihttp://dcollection.snu.ac.kr/common/orgView/000000158710ko_KR
dc.description학위논문(석사)--서울대학교 대학원 :국제농업기술대학원 국제농업기술학과,2020. 2. 강상기.-
dc.description.abstractFoot-and-Mouth Disease (FMD) is a highly infectious disease of cloven-hoofed animals like cattle, sheep, and pigs in the livestock industry causing serious loss of productivity and large-scale economic damage. Although vaccination is the reliable solution to prevent FMD, FMD is still hard to control and prevent because of a variety of subtypes and high mutation in the FMD virus. Recombinant protein vaccine is one of the good alternatives to the attenuated viral vaccine in many ways including safety issues, which deals with no virus for vaccine production. 5BT is composed by five B cell epitopes (amino acid residues 130 to 160) derived from GH loop in VP1 from the various O type FMDV and one T cell epitope (amino acid residues 21 to 350) of 3A, one of the non-structural proteins (NSPs), in FMDV.
The production of the 5BT protein from E. coli BL21 (DE3) was optimized by determination of proper IPTG induction and purification conditions. The optimal conditions for the IPTG induction and His-Tag affinity purification were achieved with 1.0mM IPTG treated at OD600 0.5 for 4 hours and subsequent 3 time-washing by 20, 30, and 50mM imidazole after binding the sample to the resin, respectively. The 5BT could be obtained as soluble proteins with 52.89mg/L yield and 95% of purity by the condition optimization.
To evaluate the efficiency of 5BT protein as FMD vaccine, we compared the serum antibody induction levels of 5BT protein with commercial vaccine (type O + type A). 8-week-old SD rats were immunized by intramuscular administration with 5BT protein and commercial vaccine in different dose levels, with 50, 100, 150, 200, or 250μg, of 5BT protein, with 20, 40, 60, 80, or 100μl, of commercial vaccine, respectively. At the 6-week after initiation of immunization, the group treated with 200μg of 5BT protein showed the highest serum IgG level among the various dose group for 5BT protein. The commercial vaccine showed the equivalent level of serum IgG with 200μg of 5BT even in the group treated with 20μl.
Usually, the adjuvants help 5BT protein antigen to enhance the immunogenicity. We also optimized the vaccine with the different adjuvants with 8 groups of SD-rats, the 200μg of 5BT but with different tendencies-treated groups in combination with various adjuvants such as CFA/IFA, Alum, MF59, MPLA, and AS04 (MPLA+Alum) were compared with a 20μl of commercial FMD ( type O+type A)-treated group and a 50μg of 5BT-treated group, additionally. After vaccination, only the group of CFA/IFA with 200μg of 5BT protein showed the early antibody induction on the 2nd week. After 6 week-vaccination, the IgG level in the group of 20μl of commercial vaccine was similar with the group with MF59. To validate the immunization efficacy of 5BT protein antigen as FMD vaccine, the levels of FMDV serotype O specific antibodies in serum were analyzed. Our 5BT protein groups with CFA/IFA and MF59 showed higher immunization efficacy than the commercial vaccine.
All effective vaccines induce inflammatory responses. In this study, all of the vaccines showed a balanced induction between humoral and cellular immune response. The commercial vaccine showed the tendency of Th2 type immune response with dominant induction of IL-4 and IgG1. However, 5BT alone, 5BT with CFA/IFA and 5BT with MF59 groups showed the tendency of Th1 type with dominant induction of IFN-γ and IgG2a.
In conclusion, the recombinant 5BT protein were produced in E. coli host as a soluble form efficiently with high yield and purity and immunization results showed the balanced induction between humoral and cellular immune responses. Therefore, recombinant 5BT protein has considered as having a potential to be as an FMD vaccine to control and prevent FMD.
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dc.description.abstractFMD (구제역)는 가축 산업에서 가축, 양 및 돼지와 같은 발굽이 달린 동물의 전염병으로 심각한 생산성 손실과 대규모 경제 피해를 초래한다. 예방 접종이 FMD를 예방하는 신뢰할 수 있는 솔루션이지만 FMD 바이러스의 다양한 하위 유형과 높은 돌연변이로 인해 FMD를 제어하고 예방하기는 여전히 어렵다. 재조합 단백질 백신은 백신 생산을 위한 바이러스가 없는 안전 문제를 포함하여 여러 방식으로 약독화된 바이러스 백신에 대한 좋은 대안 중 하나이다. 5BT는 다양한 O형 FMDV로부터 VP1의 GH루프로부터 유래된 5개의 B세포 에피토프 (아미노산 잔기 130 내지 160개) 및 NSP 중 하나인 3A의 1 개의 T세포 에피토프 (아미노산 잔기 21 내지 350개)로 구성된다. FMDV.
적절한 IPTG 유도 및 정제 조건의 결정에 의해 대장균 BL21 (DE3)로부터 5BT 단백질의 생산을 최적화하였다. IPTG 유도 및 히스티딘-태그 친화도 정제를 위한 최적 조건은 OD600 0.5에서 4 시간 동안 1.0mM IPTG로 처리한 후, 샘플을 수지에 결합시킨 후 각각 20, 30 및 50mM 이미다졸로 3 회 세척하여 달성하였다. 5BT는 조건 최적화에 의해 52,89mg/L의 수율 및 95%의 순도를 갖는 가용성 단백질로써 수득 되었다.
FMD 백신으로써 5BT 단백질의 효율을 평가하기 위해, 5BT 단백질의 혈청 항체 유도 수준을 상용 백신 (타입 O + 타입 A)과 비교하였다. 20, 40, 60, 80 및 100μl의 상용 구제역 백신, 또는 50, 100, 150, 200, 및 250μg의 상이한 용량의 5BT 단백질을 8주령의 SD-rat의 근육 내에 투여함으로써 면역반응을 유도하였다. 면역화 개시 후 6주 째에, 5BT 단백질에 대한 다양한 투여 그룹 중에서 200μg으로 처리된 그룹이 가장 높은 혈청 IgG 수준을 나타냈다. 상용 백신은 20μl로 처리된 최적 그룹에서도 200μg의 5BT와 동등한 수준의 혈청 IgG를 나타냈다.
일반적으로 어쥬번트는 백신 내에 포함되어 있는 항원의 면역원성을 향상시키는 것을 돕는다. 본 연구에서는 CFA/IFA와 같은 실험실용 어쥬번트 외에 5BT 단백질 항원과 Alum, MF59, MPLA와 같은 다양한 상용 어쥬번트와의 조합을 통해 5BT 단백질 항원의 구제역에 대한 아단위 백신으로써의 효능 향상 여부를 SD-rat을 이용한 면역시험을 통하여 평가하였다. 그 결과, 5BT와 MF59의 조합은 CFA/IFA와의 조합이나 상용 구제역 백신과 유사한 백신 효과를 나타내었다. 다음으로는 구제역백신으로써 5BT 단백질 항원의 중화항체 형성능력을 평가하기 의하여 O혈청형의 특이적 FMDV 항원을 포함하는 상용 구제역 검정 키트를 활용하여 각각의 항혈청 내의 항원 특이적 IgG 수준을 조사하였는데, 5BT와 MF59의 조합은 앞서 결과와 일치하게 상용 구제역 백신보다 높은 중화항체 형성능력을 보였다,
본 연구에서 5BT와 다영한 어쥬번트의 조합물은 대체로 체액성 면역과 세포성 면역 간에 균형 잡힌 반응을 보여주었으나, 상용 구제역 백신의 경우 IL-4 및 IgG1의 발현이 우세한 Th2 유형의 면역반응을 나타낸 반면, 5BT와 MF59의 조합의 경우에는 IFN-γ 및 IgG2a의 발현이 우세한 Th1 유형의 면역반응을 나타내었다.
결론적으로, 재조합 5BT 단백질은 가용성 형태의 높은 수율 및 순도로 대장균 숙주에서 생산 가능하며, MF59과 같은 상용 어쥬번트와의 조합을 통해 SD-rat 모델레서 상용 구제역 백신을 상회하는 중화항체 형성능력을 보여주었다. 따라서 5BT 단백질 항원은 향 후 효과적이고 경제적인 아단위 구제역 백신으로써 활용될 잠재력이 높은 것으로 평가되었다.
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dc.description.tableofcontentsLiterature Review 1
1. Foot-and-Mouth Disease 1
1.1 Foot-and-Mouth Disease Virus 1
1.1.1 FMDV genome 1
1.1.2 FMDV structure 2
1.2 FMDV variants and ecology 7
1.3 FMDV clinical symptoms 7
1.4 FMDV diagnostic methods and control 8
2. FMDV Vaccine 8
2.1 Live attenuated vaccine 8
2.2 Inactivated vaccine 9
2.3 Recombinant subunit vaccine 10
3. Solubility of recombinant proteins 11
3.1 Inclusion bodies 12
3.2 The environment of medium factors 14
3.2.1 Induction time affects the expression of recombinant protein 15
3.2.2 Inducer concentration affects the expression of recombinant protein 16
3.2.3 Incubation temperature after induction and harvest time affect the expression of recombinant protein 16
4. Adjuvant system 18
4.1 Commercial adjuvants 18
4.2 Artificial synthetic adjuvants 20

Chapter 1. Optimization for high yield of target protein, 5BT 22
1. Introduction 22
2. Materials and Methods 26
2.1 Production of proteins 26
2.1.1 Expression of 5BT protein 26
2.1.2 Characterization of recombinant target protein 26
2.1.2.1 Optimization of culture conditions to improve 5BT protein production 26
2.1.2.2 Analysis of protein expression and protein solubility 27
2.2 Optimization of purification condition to improve the purity of 5BT protein 28
2.2.1 Ion exchange chromatography 28
2.2.2 Optimization of His-Tag affinity purification 28
2.2.3 Protein dialysis and freeze drying 29
2.2.4 Quantification of recombinant target proteins 29
3. Results and discussion 32
3.1 Confirmation of the target gene and protein expression 32
3.2 Optimization of the 5BT protein expression condition 36
3.2.1 The solubility of recombinant protein 36
3.2.2 Quantification of recombinant proteins 37
3.3 Optimization of purification condition 42
3.3.1 Ion exchange purification 42
3.2.2 Ni-NTA His-Tag affinity chromatography 43
4. Conclusion 47

Chapter 2. In vivo evaluation of 5BT protein for the FMD subunit vaccine 49
1. Introduction 49
2. Materials and methods 51
2.1 SD-rat immunization and blood sampling 51
2.2 The plans of immunization in SD-rat 51
2.2.1 1st animal experiment: Investigation of dose-dependent immune response of 5BT 52
2.2.2 2nd animal experiment: Investigation of dose-dependent immune response of the commercial FMD vaccine 54
2.2.3 3rd animal experiment: Exploration of effective adjuvants for 5BT 55
2.3 Serological analysis 56
2.3.1 Determination of 5BT-specific serum IgG by indirect ELISA 56
2.3.2 Determination of type O FMDV-specific serum IgG by FMDV ELISA kit 57
2.3.3 Cytokine ELISA 58
2.4 Statistical analysis 58
3. Results and Discussion 59
3.1 Immunogenicity evaluation of 5BT protein as an FMD vaccine 59
3.1.1 Dose-dependent immunogenicity of 5BT in rat model 59
3.1.2 Dose-dependent immunogenicity of commercial FMD vaccine in rat model 63
3.1.3 Selection of effective adjuvant for 5BT 66
3.2 Efficacy validation of 5BT as an FMD vaccine 72
4. Conclusion 77

Overall conclusion 80
Literature Cited 83
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dc.language.isoeng-
dc.publisher서울대학교 대학원-
dc.subject.ddc631-
dc.titleEfficiency Evaluation of 5BT, a Genetically Recombinant Multi-epitope-protein, for Its Application as a Subunit Vaccine against Foot and Mouth Disease-
dc.title.alternative유전자 재조합형 다중 항원결정기 단백질, 5BT의 구제역 대응 아단위 백신으로써의 활용을 위한 효율성 평가-
dc.typeThesis-
dc.typeDissertation-
dc.contributor.AlternativeAuthor호리나-
dc.contributor.department국제농업기술대학원 국제농업기술학과-
dc.description.degreeMaster-
dc.date.awarded2020-02-
dc.contributor.major경제동물산업기술-
dc.identifier.uciI804:11032-000000158710-
dc.identifier.holdings000000000042▲000000000044▲000000158710▲-
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