Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
8360054 | Protein Expression and Purification | 2015 | 6 Pages |
Abstract
Interleukin-33 (IL-33) is a member of the IL-1 family and the ligand of orphan ST2 molecules. IL-33 is widely expressed in multiple tissues and cells, and mainly involved in regulating Th2 immune and inflammatory responses. Inhibiting IL-33 signaling pathways relieves the symptoms of allergic inflammation, indicating that IL-33 is a potential target for the treatment of allergic diseases. In this study, the recombinant vectors SP-scFv-Fc/pcDNA3.1 and SP-scFv-Fc/PMH3EN were constructed to express a human scFv-Fcs against IL-33. The size of the inserted SP-scFv-Fc was approximately 1540Â bp. The RT-PCR results showed that SP-scFv-Fcs were successfully transfected into CHO K1 cells. Western blot analysis indicated specific binding of the expressed scFv-Fcs fusion protein (approximately 60Â kDa under reduced condition) with a goat anti-human IgG1 Fc antibody. The expression level of the scFv-Fcs from SP-scFv-Fc/PMH3EN was higher than that from SP-scFv-Fc/pcDNA3.1. A single high-expressing cell line was selected after three rounds of screening and the fusion protein was expressed in a suspension culture in serum-free medium. The level of expression products reached 20Â mg/L and the expressed and purified scFvs was further characterized and analyzed for bioactivity and functionality. The recombinant vectors for eukaryotic expression of scFv-Fcs against IL-33 were successfully constructed and the expressed scFv-Fcs was shown to be a suitable candidate for the development of a new therapy for allergic and autoimmune diseases.
Keywords
Related Topics
Life Sciences
Biochemistry, Genetics and Molecular Biology
Biochemistry
Authors
Yingchun Ye, Siji Nian, Wenfeng Xu, Tong Wu, Xu Wang, Yan Gao, Qing Yuan,