Construction and expression of human scFv-Fc against interleukin-33.

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/PMH3(EN) were constructed to express a human scFv-Fcs against IL-33. The size of the inserted SP-scFv-Fc was approximately 1540bp. 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 60kDa under reduced condition) with a goat anti-human IgG1 Fc antibody. The expression level of the scFv-Fcs from SP-scFv-Fc/PMH3(EN) 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 20mg/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.