Recombinant Soybean Protein β-Conglycinin α′-Subunit Expression and Induced Hypersensitivity Reaction in Rats

Abstract

Background: The major storage protein in soybean seed is β-conglycinin and this protein has been identified as being responsible for food-allergic reactions in several species. However, the mechanism through which β-conglycinin induces an allergic reaction has not yet been elucidated. In addition, assessing the antigenic activity of β-conglycinin by studying the activity of a subunit has rarely been conducted. Therefore, the objective of the present study was to characterize the antigenic specificity of the β-conglycinin α′-subunit. Methods: We established an Escherichia coli expression system to obtain β-conglycinin α′-subunit. The fusion proteins were then used in a rat model to induce a hypersensitive reaction. Immunoblotting, IgE and IgG1 level, histamine release, and passive cutaneous anaphylaxis reactions and intestinal histology were tested to assess the allergenic activity of the β-conglycinin α′-subunit. Results: Pure β-conglycinin α′-subunit was obtained by expression in E. coli. The recombinant proteins were shown to have the same biological activity as the natural β-conglycinin α′-subunit using immunoblotting analysis. Both the IgE and IgG1 level in serum and the histamine concentration in the intestine were increased while passive cutaneous anaphylactic reactions were induced in Brown Norway rats by intragastric gavage with the α′-subunit. Histamine release of mast cells was also elevated in vitro. Conclusions: Our results indicate that the β-conglycinin α′-subunit possesses an intrinsic immune-stimulating capacity and that it can induce an allergic reaction. Moreover, this study showed that β-conglycinin α′-subunit-induced anaphylaxis is IgE mediated, and mast cell degranulation and histamine release are associated with anaphylactic symptoms.

Expression of the soybean allergenic protein P34 in Escherichia coli and its indirect ELISA detection method

“…Moreover, it is incredibly time consuming and depends on expensive equipment. In comparison, prokaryotic recombinant protein expression systems (E. coli) have several advantages, including ease of culture, rapid cell growth, and relatively simple purification procedures (Bohle and Vieths 2004;Clavijo et al 2004;Guo et al 2008;Lorenz et al 2001;Utsumi et al 2002). In addition, immunoblot analysis showed that both soybean and recombinant P34 proteins cross-reacted not only with polyclonal and monoclonal antibodies produced against P34 and crude soybean protein but also with the sera from 361 patients with atopic dermatitis; it means that the recombinant P34 is immunologically reactive, indicating that the recombinant P34 expressed by E. coli has similar epitope structures to natural soybean P34 protein and thus can be used as the standard allergen of P34 (Babiker et al 2000).…”

Bioelimination of histamine in epithelia of the porcine proximal colon of pigs

“…In recent years, bioelimination of histamine has attracted considerable interest because inadequate bioavailability of histamine has been linked to a number of pathological conditions [2,[8][9][10][11]. The current understanding of histamine bioelimination distinguishes between local and systemic components.…”

“…In pathological conditions like allergy, asthma, parasitic disease or mastocytosis, very high amounts of histamine can be released from mast cells and basophils [2,[8][9][10][11]. When the capacity for local histamine elimination is overtaxed, the amine spills over into the systemic circulation and can give rise to deleterious elevations in plasma histamine level [12,13].…”

Low immunoreactive glycated soybean antigen proteins production: system-wide analysis of their immunogenicityin vitroandin vivo

“…Protein content was determined by the Kjeldahl method and purity was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using mini-gel apparatus (BioRad Laboratories, CA, America) followed by Coomassie Brilliant Blue R-250 staining. The gel was scanned by Bio Imaging System with Gene Genius (Syngene, USA), and the purity of glycinin was measured by GeneTools Analysis Software, Version 3.03.03 (Guo et al, 2008). Glycinin and β-conglycinin were glycated with FOS according to the Maillard reaction in powder system described by Jürgen et al (2007).…”

“…Soybean is considered to be one of the major food allergens, which is more prominent in the industrial countries (Wang, Qin, Sun, & Zhao, 2014). In recent years, soybean allergy has become a public problem all over the world (Guo, Piao, Cao, & Ou, 2008;Wang et al, 2014). Glycinin and β-conglycinin are two major antigen proteins in soybean and they accounted for about 65-80% of the total seed protein.…”