Generation of the IgCw-γ1ε2-4/κ composed of the constant domain Cγ1 and Cε2-4
- 주제(키워드) Immunoglobulin E , constant region , Cε , isotype control , Reference antibody
- 발행기관 아주대학교
- 지도교수 권명희
- 발행년도 2021
- 학위수여년월 2021. 2
- 학위명 석사
- 학과 및 전공 일반대학원 의생명과학과
- 실제URI http://www.dcollection.net/handler/ajou/000000030507
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
In this study, a novel recombinant antibody fragment IgCw-γ1ε2-4/κ (IgCw-γε/κ) was constructed and evaluated for its potential as an alternative isotype control, which can be used in place of IgE. IgCw-γε/κ (~140 kDa) is composed of two hybrid human constant heavy chains (Cγ1-hinge region-Cε2-4) and two human constant kappa light chains (Cκ). IgCw-γε/κ was expressed as an assembled form of the heavy and light chains, and the production yield of IgCw-γε/κ obtained from a culture of transfected HEK293F cells was 17 mg/L, which is comparable to that of full-size IgE. Binding of IgCw-γε/κ to human Fc epsilon receptor Ⅰ (FcεRⅠ) expressed on RBL-2H3-hFcεRⅠα cells was confirmed using flow cytometry. A standard curve generated using IgCw-γε/κ was used for the measurement of IgE concentrations. Further, the sensitization with IgCw-γε/κ of RBL-2H3-hFcεRⅠα cells and subsequent stimulation with anti-human kappa light chain antibody induced β-hexosaminidase release. IgCw-γε/κ also acted as an FcεRⅠ blocker to suppress IgE-FcεRⅠ interaction. Taken together, these results demonstrate that IgCw-γε/κ can be used as a functional alternative isotype control, instead of full-size IgE, in biomedical research fields.
more목차
Ⅰ. INTRODUCTION 1
Ⅱ. MATERIALS AND METHODS 6
A. Construction of plasmid vectors 6
B. Purification of proteins 16
C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining 16
D. Immunoblotting 17
E. Size-exclusion chromatography (SEC) 17
F. Cell culture 18
G. Establishment of stable cell line 18
H. Flow cytometry 19
I. Enzyme-Linked Immunosorbent assay (ELISA) 20
J. β-hexosaminidase release assay 21
Ⅲ. RESULT 22
A. Production of IgCw-γε/κ using HEK293F cells 22
B. Binding of IgCw-γε/κ to human FcεRⅠα chain expressed on RBL-2H3-cell surface 30
C. Quantitative determination of IgE concentrations using IgCw-γε/κ 34
D. Release of β-hexosaminidase and inhibition of IgE-FcεRⅠ binding by IgCw-γε/κ 37
Ⅳ. DISCUSSION 40
Ⅴ. CONCLUSION 43
REFERENCES 44
국문요약 51
