title: 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.
authors.affiliation: Department of Basic Medicine, Luzhou Medical College, Luzhou, Sichuan Province 646000, China. Electronic address: 351584908@qq.com.
authors.forename: Yingchun
authors.initials: Y
authors.lastname: Ye
authors.affiliation: Department of Basic Medicine, Luzhou Medical College, Luzhou, Sichuan Province 646000, China. Electronic address: sijinian@hotmail.com.
authors.forename: Siji
authors.initials: S
authors.lastname: Nian
authors.affiliation: Department of Basic Medicine, Luzhou Medical College, Luzhou, Sichuan Province 646000, China. Electronic address: x.wenfeng@yahoo.com.
authors.forename: Wenfeng
authors.initials: W
authors.lastname: Xu
authors.affiliation: Department of Basic Medicine, Luzhou Medical College, Luzhou, Sichuan Province 646000, China. Electronic address: 7631046@qq.com.
authors.forename: Tong
authors.initials: T
authors.lastname: Wu
authors.affiliation: Department of Basic Medicine, Luzhou Medical College, Luzhou, Sichuan Province 646000, China. Electronic address: wangxu322@163.com.
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Basic Medicine, Luzhou Medical College, Luzhou, Sichuan Province 646000, China. Electronic address: 747094571@qq.com.
authors.forename: Yan
authors.initials: Y
authors.lastname: Gao
authors.affiliation: Department of Basic Medicine, Luzhou Medical College, Luzhou, Sichuan Province 646000, China. Electronic address: yuanqing_ok@yahoo.com.
authors.forename: Qing
authors.initials: Q
authors.lastname: Yuan
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000067596:Interleukin-33 / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
chemical_list: D007141:Immunoglobulin Fc Fragments; D000067596:Interleukin-33; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies
keywords: Eukaryotic expression vector; Human IL-33; Human scFv-Fc