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Validation of particulate TLR agonists as vaccine adjuvants for immunization efficiency of M5BT, multi-epitope subunit vaccine : 구제역 바이러스 방어를 위한 멀티-에피톱 아단위 백신 M5BT의 면역 증강제로서 미립자화 TLR 아고니스트의 효과 검증

DC Field Value Language
dc.contributor.advisor최윤재-
dc.contributor.author정어진-
dc.date.accessioned2017-07-14T06:48:29Z-
dc.date.available2020-04-01T02:21:28Z-
dc.date.issued2017-02-
dc.identifier.other000000141063-
dc.identifier.urihttps://hdl.handle.net/10371/125988-
dc.description학위논문 (석사)-- 서울대학교 대학원 : 농생명공학부, 2017. 2. 최윤재.-
dc.description.abstractMulti-epitope vaccine is one of the strategies to cope with Foot-and-mouth disease which has significant global impact. However, low immunogenicity derived from its protein structure requires aids of adjuvant. CpG ODN and poly I:C are agonists recognized by certain toll-like receptor expressed on immune cells and thereby, modulate innate and adaptive immunity.
In this study, first, modified medium was suggested for vaccine production. It was confirmed E.coli cultured in modified terrific broth-2 (T2B) showed improved cell and protein yield.
Next, the synergistic adjuvant effect of CpG ODN and poly I:C was evaluated by cell viability, cytokine release and antigen-specific T and B cell immune response through particle formation between vaccine and adjuvant via ionic interaction. Consequently, the adjuvanticity of two TLR agonists had positive effects on cell proliferation, pro-and anti-inflammatory cytokine increase and elicited desirable T and B cell immunity.
Therefore, this study was suggested as advanced subunit vaccine strategy combined with efficient culture system and adjuvant partner against FMD infection.		-
dc.description.tableofcontentsI. Introduction 1

II. Review of Literature 3
1. FMD and vaccine 3
1) FMD 3
2) FMDV 5
3) Vaccine strategies against FMDV 7
4) Multi-epitope vaccine for FMD 10
2. Adjuvant system 12
1) Vaccine adjuvant 12
(1) Carrier adjuvant 15
(2) Immunostimulatory adjuvant 17
3. TLR agonist 19
1) TLR 19
2) CpG ODN 22
3) poly I:C 26
4) TLR synergy 29

III. Materials and Methods 31
1. Preparation of protein 31
1) Strain, plasmid and protein 31
2) Medium used for E.coli growth 33
(1) Growth curve 35
(2) Relative expression of M5BT 35
3) Ni-NTA affinity chromatography 35
4) Quantification of M5BT 38
5) Endotoxin removal 38
2. In vitro validation of TLR synergy 39
1) Cell line 39
2) TLR agonists and reagents 39
3) In vitro stability 40
4) Cell viability 40
5) mRNA expression 41
(1) Primer design 41
(2) Preparation of RNA and qRT-PCR 42
6) BMDC generation 43
(1) Mice and reagents 43
(2) Isolation of BM cell and culture 43
(3) Validation of BMDC 44
(4) TLR expression 45
7) Cytokine ELISA 47
3. Particle formation of M5BT with TLR agonists 47
1) Particle formulation via ionic interaction 47
2) Morphology of particle 47
4. In vivo immunization in mouse 49
1) Mice and immunization scheme 49
2) Spleen isolation and cytokine ELISA 50
3) Antibody response 50
(1) PI test 50
(2) M5BT-specific ELISA 51
5. Statistical analysis 52

IV. Results and discussion 53
1. Development of optimal medium for M5BT protein production in E.coli 53
1) Effect of medium composition on E.coli growth 53
2) Relative expression of soluble M5BT protein 55
3) Quantification of purified M5BT 57
2. In vitro adjuvanticity validation of TLR agonists 60
1) In vitro stability and cell viability 60
2) mRNA expression of pro- anti-inflammatory cytokine in Raw264.7 cell 64
3) Th1 response related cytokine ELISA 67
3. Particle formation of M5BT with TLR agonists 70
4. In vivo mouse immunization 73
1) M5BT-specific ELISA 73
2) Cytokine ELISA 76

V. Conclusion and Further Prospects 79

VI. Literature Cited 81

VII. Summary in Korean 94		-
dc.formatapplication/pdf-
dc.format.extent2417606 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subjectFoot-and-mouth disease-
dc.subjectAdjuvant-
dc.subjectMulti-epitope vaccine-
dc.subjectTLR agonist-
dc.subjectCpG ODN-
dc.subjectpoly I:C-
dc.subjectsynergistic effect-
dc.subject.ddc630-
dc.titleValidation of particulate TLR agonists as vaccine adjuvants for immunization efficiency of M5BT, multi-epitope subunit vaccine-
dc.title.alternative구제역 바이러스 방어를 위한 멀티-에피톱 아단위 백신 M5BT의 면역 증강제로서 미립자화 TLR 아고니스트의 효과 검증-
dc.typeThesis-
dc.contributor.AlternativeAuthorJung, Eojin-
dc.description.degreeMaster-
dc.citation.pages94-
dc.contributor.affiliation농업생명과학대학 농생명공학부-
dc.date.awarded2017-02-
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