3D8 항체의 상보성 결정 부위 변이가 3D8 항체의 활성에 미치는 영향
Effect of disruption of 3D8 complementarity-determining regions on properties of 3D8 antibody
- 주제(키워드) Complementarity-Determining Region (CDR) , 3D8 single chain variable fragment (scFv) , DNA-binding activity , DNA-hydrolyzing activity , Cell-penetrating activity
- 발행기관 아주대학교
- 지도교수 권명희
- 발행년도 2012
- 학위수여년월 2012. 8
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000012703
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Modifications such as substitution, insertion, or deletion of amino acids within or near CDRs (complementarity-determining regions) in antibodies have been utilized to improve antigen-binding affinity and/or stability. However, little is reported about functional consequence by the disruption of whole amino acid sequence in CDRs of an antibody with multiple activities. Here, we dissected influence of each whole CDR-disruption on the function of an antibody, 3D8 scFv (single chain variable fragment; variable region of heavy chain connected to variable region of light chain by (G4S1)3 linker) which has DNA-binding/-hydrolyzing, heparin-binding and cell-penetrating activities. We generated single CDR-disrupted antibodies in the formats of scFv (sc-H/LCDRi; inactivated-CDR of variable domain of heavy or light chain in scFv format), VH (H-CDRi; inactivated-CDR of variable domain of heavy chain single domain format), and VL (L-CDRi; inactivated-CDR of variable domain of light chain single domain format) by replacing the amino acid sequence of each CDRs with (GlySer)n of same length. DNA-binding activity of sc-variants except sc-L1i was similar to 3D8 scFv, and VH variants had higher affinity to DNA than VH wild type. Therefore, L-CDR1 in 3D8 scFv format is likely to be responsible for DNA-binding activity. Sc-H3i and sc-L3i still retained the heparin-binding activity similar to 3D8 scFv. Moreover, sc-H3i, sc-L3i and H-3i entered the HeLa cells and localized in cytosol of the HeLa cells like 3D8 scFv. It seems that at least H-CDR3 and L-CDR3 in 3D8 scFv format contribute to interaction with heparin and penetration the HeLa cells. For DNA-hydrolyzing activity, sc-L1i, sc-L3i, 3D8 VH and VH’s variants did not hydrolyze DNA. Thus L-CDR1 and L-CDR3 in 3D8 scFv format is not related with hydrolysis of DNA. Next, we examined which CDRs are responsible for the activities of 3D8 antibody using synthesized peptides corresponding to 3D8-CDRs. Only pep-L1 bound to DNA or heparin, however pep-L1 did not enter the HeLa cells. Pep-H1 and pep-H2 entered the HeLa cells, even though much lower penetration efficiency than Tat peptide. Consequently, our results show that effect of the single CDR-disruptions on activities of 3D8 antibody is different between scFv format and single domain format. Only L-CDR1 of 3D8 scFv is important region for DNA-binding/hydrolyzing and cell-penetrating activities.
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ABSTRACT i
TABLE OF CONTENTS ii
LIST OF FIGURES iii
LIST OF TABLES iv
ABBREVIATION v
I. INTRODUCTION 1
II. MATERIALS AND METHODS 7
A. Peptides 7
B. Plasmid constructions 7
C. Cell culture 7
D. Bacterial expression and purification of 3D8 variants 8
E. Sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) 9
F. Enzyme-linked immunosorbent assay (ELISA) 9
1) Biotin-labeled peptides 9
2) 3D8 variants 10
G. Competitive ELISA 10
H. Surface plasmon resonance (SPR) 11
I. Flow cytometry 12
1) FITC-labeled peptides 12
2) 3D8 variants 13
J. Confocal laser scanning microscopy 14
1) FITC-labeled peptides 14
2) 3D8 CDR variants 14
K. DNA/RNA-hydrolyzing activity 15
III. RESULTS 17
A. Bacterial expression and purification of 3D8 variants 17
B. DNA-binding activity of 3D8 variants 24
C. Heparin-binding activity of 3D8 variants 31
D. Competitive ELISA of 3D8 scFv 33
E. Cellular internalization of 3D8 variants 35
F. DNA-hydrolyzing activity of 3D8 variants 41
G. Properties of peptide corresponding to 3D8 CDRs 45
IV. DISCUSSION 50
V. CONCLUSION 53
REFERENCES 54
국문요약 60
