Differential effects of heating modes on the immunogenic potential of soy-derived peptides released after in vitro infant digestion.

During production of soy-based infant formula, soy protein undergoes heating processes. This study investigated the differential impact of heating modes on the immunogenic potential of peptides in soy protein digests. Wet or dry heating was applied, followed by in vitro gastrointestinal infant digestion. The released peptides were analyzed by LC-MS/MS. Bioinformatics tools were utilized to predict and identify potential linear B-cell and T-cell epitopes, as well as to explore cross-reactivity with other legumes. Subsequently, the peptide intensities of the same potential epitope across different experimental conditions were compared. As a result, we confirmed the previously observed enhancing effect of wet heating on infant digestion and inhibitory effect of dry heating. A total of 8,546 peptides were detected in the digests, and 6,684 peptides were with a score over 80. Among them, 29 potential T-cell epitopes and 27 potential B-cell epitopes were predicted. Cross-reactivity between soy and other legumes, including peanut, pea, chickpea, lentil, kidney bean, and lupine, was also detected. Overall, heating and digestion time could modulate the potential to trigger peptide-induced immune responses.

Effects of Chlorogenic Acid on Lowering IgE-Binding Capacity of Soybean 7S: Comparison between Covalent and Noncovalent Interaction.

Allergenicity of soybean 7S protein (7S) troubles many people around the world. However, many processing methods for lowering allergenicity is invalid. Interaction of 7S with phenolic acids, such as chlorogenic acid (CHA), to structurally modify 7S may lower the allergenicity. Hence, the effects of covalent (C-I, periodate oxidation method) and noncovalent interactions (NC-I) of 7S with CHA in different concentrations (0.3, 0.5, and 1.0 mM) on lowering 7S allergenicity were investigated in this study. The results demonstrated that C-I led to higher binding efficiency (C-0.3:28.51 ± 2.13%) than NC-I (N-0.3:22.66 ± 1.75%). The C-I decreased the α-helix content (C-1:21.06%), while the NC-I increased the random coil content (N-1:24.39%). The covalent 7S-CHA complexes of different concentrations had lower IgE binding capacity (C-0.3:37.38 ± 0.61; C-0.5:34.89 ± 0.80; C-1:35.69 ± 0.61%) compared with that of natural 7S (100%), while the noncovalent 7S-CHA complexes showed concentration-dependent inhibition of IgE binding capacity (N-0.3:57.89 ± 1.23; N-0.5:46.91 ± 1.57; N-1:40.79 ± 0.22%). Both interactions produced binding to known linear epitopes. This study provides the theoretical basis for the CHA application in soybean products to lower soybean allergenicity.

Localization of G1A1a Allergenic Domain Destroyed by Thermal Processing.

Glycinin is an important allergenic protein. A1a is the acidic chain of the G1 subunit in glycinin (G1A1a), and it has strong allergenicity. In this study, we used phage display technology to express the protein of G1A1a and its overlapping fragments and an indirect enzyme-linked immunosorbent assay (iELISA) to determine the antigenicity and allergenicity of the expressed protein. After three rounds of screening, it was determined that fragment A1a-2-B-I (151SLENQLDQMPRRFYLAGNQEQEFLKYQQEQG181) is the allergenic domain of G1A1a destroyed by thermal processing. In addition, three overlapping peptides were synthesized from fragments A1a-2-B-I, and a linear epitope was found in this domain through methods including dot blot and iELISA. Peptide 2 (157DQMPRRFYLANGNQE170) showed allergenicity, and after replacing it with alanine, it was found that amino acids D157, Q158, M159, and Y164 were the key amino acids that affected its antigenicity, while Q158, M159, R162, and N168 affected allergenicity.

Conformation-Activity Mechanism of Alcalase Hydrolysis for Reducing In Vitro Allergenicity of Instant Soy Milk Powder.

Effective reduction of the allergenicity of instant soy milk powder (ISMP) is practically valuable for expanding its applications. This study optimized the enzymolysis technology of ISMP using single-factor experiments and response surface methodology, combined serological analysis, cellular immunological models, bioinformatics tools, and multiple spectroscopy techniques to investigate the effects of alcalase hydrolysis on allergenicity, spatial conformation, and linear epitopes of ISMP. Under the optimal process, special IgE and IgG1 binding abilities and allergenic activity to induce cell degranulation of alcalase-hydrolyzed ISMP were reduced by (64.72 ± 1.76)%, (56.79 ± 3.72)%, and (73.3 ± 1.19)%, respectively (P < 0.05). Moreover, the spatial conformation of instant soy milk powder hydrolysates (ISMPH) changed, including decreased surface hydrophobicity, a weaker peak of amide II band, lower contents of α-helix and ß-sheet, and an enhanced content of random coil. Furthermore, the linear epitopes of major soy allergens, 9 from glycinin and 13 from ß-conglycinin, could be directionally disrupted by alcalase hydrolysis. Overall, the structure-activity mechanism of alcalase hydrolysis to reduce ISMP allergenicity in vitro was preliminarily clarified. It provided a new research direction for the breakthrough in the desensitization of ISMP and a theoretical basis for revealing the potential mechanism of alcalase enzymolysis to reduce the allergenicity of ISMP.

Silencing Autophagy-Related Gene 2 (ATG2) Results in Accelerated Senescence and Enhanced Immunity in Soybean.

Autophagy plays a critical role in nutrient recycling and stress adaptations. However, the role of autophagy has not been extensively investigated in crop plants. In this study, soybean autophagy-related gene 2 (GmATG2) was silenced, using virus-induced silencing (VIGS) mediated by Bean pod mottle virus (BPMV). An accelerated senescence phenotype was exclusively observed for the GmATG2-silenced plants under dark conditions. In addition, significantly increased accumulation of both ROS and SA as well as a significantly induced expression of the pathogenesis-related gene 1 (PR1) were also observed on the leaves of the GmATG2-silenced plants, indicating an activated immune response. Consistent with this, GmATG2-silenced plants exhibited a significantly enhanced resistance to Pseudomonas syringae pv. glycinea (Psg) relative to empty vector control plants (BPMV-0). Notably, the activated immunity of the GmATG2-silenced plants was independent of the MAPK signaling pathway. The fact that the accumulation levels of ATG8 protein and poly-ubiquitinated proteins were significantly increased in the dark-treated GmATG2-silenced plants relative to the BPMV-0 plants indicated that the autophagic degradation is compromised in the GmATG2-silenced plants. Together, our results indicated that silencing GmATG2 compromises the autophagy pathway, and the autophagy pathway is conserved in different plant species.

Intestinal Transcriptome Analysis Reveals Enrichment of Genes Associated with Immune and Lipid Mechanisms, Favoring Soybean Meal Tolerance in High-Growth Zebrafish (Danio Rerio).

The molecular mechanisms underlying fish tolerance to soybean meal (SBM) remain unclear. Identifying these mechanisms would be beneficial, as this trait favors growth. Two fish replicates from 19 experimental families were fed fishmeal-(100FM) or SBM-based diets supplemented with saponin (50SBM + 2SPN) from juvenile to adult stages. Individuals were selected from families with a genotype-by-environment interaction higher (HG-50SBM + 2SPN, 170 ± 18 mg) or lower (LG-50SBM + 2SPN, 76 ± 10 mg) weight gain on 50SBM + 2SPN for intestinal transcriptomic analysis. A histological evaluation confirmed middle intestinal inflammation in the LG- vs. HG-50SBM + 2SPN group. Enrichment analysis of 665 differentially expressed genes (DEGs) identified pathways associated with immunity and lipid metabolism. Genes linked to intestinal immunity were downregulated in HG fish (mpx, cxcr3.2, cftr, irg1l, itln2, sgk1, nup61l, il22), likely dampening inflammatory responses. Conversely, genes involved in retinol signaling were upregulated (rbp4, stra6, nr2f5), potentially favoring growth by suppressing insulin responses. Genes associated with lipid metabolism were upregulated, including key components of the SREBP (mbtps1, elov5l, elov6l) and cholesterol catabolism (cyp46a1), as well as the downregulation of cyp7a1. These results strongly suggest that transcriptomic changes in lipid metabolism mediate SBM tolerance. Genotypic variations in DEGs may become biomarkers for improving early selection of fish tolerant to SMB or others plant-based diets.

Predicting the allergenicity of legume proteins using a PBMC gene expression assay.

BACKGROUND: Food proteins differ in their allergenic potential. Currently, there is no predictive and validated bio-assay to evaluate the allergenicity of novel food proteins. The objective of this study was to investigate the potential of a human peripheral blood mononuclear cell (PBMC) gene expression assay to identify biomarkers to predict the allergenicity of legume proteins. RESULTS: PBMCs from healthy donors were exposed to weakly and strongly allergenic legume proteins (2S albumins, and 7S and 11S globulins from white bean, soybean, peanut, pea and lupine) in three experiments. Possible biomarkers for allergenicity were investigated by exposing PBMCs to a protein pair of weakly (white bean) and strongly allergenic (soybean) 7S globulins in a pilot experiment. Gene expression was measured by RNA-sequencing and differentially expressed genes were selected as biomarkers. 153 genes were identified as having significantly different expression levels to the 7S globulin of white bean compared to soybean. Inclusion of multiple protein pairs from 2S albumins (lupine and peanut) and 7S globulins (white bean and soybean) in a larger study, led to the selection of CCL2, CCL7, and RASD2 as biomarkers to distinguish weakly from strongly allergenic proteins. The relevance of these three biomarkers was confirmed by qPCR when PBMCs were exposed to a larger panel of weakly and strongly allergenic legume proteins (2S albumins, and 7S and 11S globulins from white bean, soybean, peanut, pea and lupine). CONCLUSIONS: The PBMC gene expression assay can potentially distinguish weakly from strongly allergenic legume proteins within a protein family, though it will be challenging to develop a generic method for all protein families from plant and animal sources. Graded responses within a protein family might be of more value in allergenicity prediction instead of a yes or no classification.

Lunasin Peptide is a Modulator of the Immune Response in the Human Gastrointestinal Tract.

INTRODUCTION: Lunasin is a soybean bioactive peptide with a variety of beneficial properties against chronic disorders. However, its effect in human primary intestinal cells remains unknown. Hence, this study aims to characterize its ex vivo biological activity in the human intestinal mucosa. METHODS AND RESULTS: Human intestinal biopsies, obtained from healthy controls, are ex vivo conditioned with lunasin both in the presence/absence of lipopolysaccharide (LPS). Peptide maintains its stability during biopsy culture by HPLC-MS/MS analysis. Lunasin is bioactive in the human mucosa, as it induces IL-1ß, TNF-α, IL-17A, CCL2, and PGE2/COX-2 gene expression together with an increased expression of tolerogenic IL-10 and TGFß, while it also downregulates the expression of iNOS and subunit p65 from NF-κB. Indeed, lunasin also abrogates the LPS-induced pro-inflammatory response, downregulating IL-17A, IFNγ, and IL-8 expression, and inducing IL-10 and TGFß expression. These results are also mirrored in the cell-free culture supernatants at the protein level by Multiplex. Moreover, lunasin further induces a regulatory phenotype and function on human intestinal conventional dendritic cell and macrophage subsets as assessed by flow cytometry. CONCLUSIONS: We hereby have characterized lunasin as an immunomodulatory peptide with potential capacity to prevent immune and inflammatory-mediated disorders in the human gastrointestinal tract.

Aptamers against the ß-Conglutin Allergen: Insights into the Behavior of the Shortest Multimeric (Intra)Molecular DNA G-Quadruplex.

In previous work, a 93-mer aptamer was selected against the anaphylactic allergen, ß-conglutin and truncated to an 11-mer, improving the affinity by two orders of magnitude, whilst maintaining the specificity. This 11-mer was observed to fold in a G-quadruplex, and preliminary results indicated the existence of a combination of monomeric and higher-order structures. Building on this previous work, in the current study, we aimed to elucidate a deeper understanding of the structural forms of this 11-mer and the effect of the structure on its binding ability. A battery of techniques including polyacrylamide gel electrophoresis, high-performance liquid chromatography in combination with electrospray ionization time-of-flight mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight, thermal binding analysis, circular dichroism and nuclear magnetic resonance were used to probe the structure of both the 11-mer and the 11-mer flanked with TT- at either the 5' or 3' end or at both ends. The TT-tail at the 5' end hinders stacking effects and effectively enforces the 11-mer to maintain a monomeric form. The 11-mer and the TT- derivatives of the 11-mer were also evaluated for their ability to bind its cognate target using microscale thermophoresis and surface plasmon resonance, and biolayer interferometry confirmed the nanomolar affinity of the 11-mer. All the techniques utilized confirmed that the 11-mer was found to exist in a combination of monomeric and higher-order structures, and that independent of the structural form present, nanomolar affinity was observed.

A novel approach to ameliorate experimental milk allergy based on the oral administration of a short soy cross-reactive peptide.

Food allergy is on the rise, and preventive/therapeutic procedures are needed. We explored a preventive protocol for milk allergy with the oral administration of a Gly-m-Bd-30K soy-derived peptide that contains cross-reactive epitopes with bovine caseins. B/T-cross-reactive epitopes were mapped using milk-specific human sera and monoclonal antibodies on overlapping and recombinant peptides of Gly-m-Bd-30K by SPOT and cell proliferation assays. Bioinformatics tools were used to characterize epitopes on the 3D-modelled molecule, and to predict the binding to HLA alleles. The peptide was orally administrated to mice that were then IgE-sensitized to milk proteins. Immunodominant B-epitopes were mainly located on the surface of the Nt-fragment. The use of a soy-peptide-containing an immunodominant cross-reactive T-epitope, along with a single B epitope, prevents IgE-mediated milk sensitization through the induction of Th1-mediated immunity and induction of blocking IgG. The use of a safe soy-peptide may represent a promising alternative for preventing milk allergy.

Effects of heat treatment on the antigenicity, antigen epitopes, and structural properties of ß-conglycinin.

In this study, the effects of heat treatment on antigenicity, antigen epitopes, and structural changes in ß-conglycinin were investigated. Results showed that the IgG (Immunoglobulin G) binding capacity of heated protein was inhibited with increased temperature, although IgE (Immunoglobulin E) binding capacity increased. Linear antigen epitopes generally remained intact during heat treatment. After heat treatment, ß-conglycinin was more easily hydrolyzed by digestive enzymes, and a large number of linear epitopes was destroyed. In addition, heat denaturation of ß-conglycinin led to the formation of protein aggregates and reduction of disulfide bonds. The contents of random coils and ß-sheet of heated ß-conglycinin decreased, but the contents of ß-turn and α-helix increased. Moreover, the protein structure of heated ß-conglycinin unfolded, more hydrophobic regions were exposed, and the tertiary structure of ß-conglycinin was destroyed. Heat treatment affected the antigenicity and potential sensitization of ß-conglycinin by changing its structure.

Local immune response to food antigens drives meal-induced abdominal pain.

Up to 20% of people worldwide develop gastrointestinal symptoms following a meal1, leading to decreased quality of life, substantial morbidity and high medical costs. Although the interest of both the scientific and lay communities in this issue has increased markedly in recent years, with the worldwide introduction of gluten-free and other diets, the underlying mechanisms of food-induced abdominal complaints remain largely unknown. Here we show that a bacterial infection and bacterial toxins can trigger an immune response that leads to the production of dietary-antigen-specific IgE antibodies in mice, which are limited to the intestine. Following subsequent oral ingestion of the respective dietary antigen, an IgE- and mast-cell-dependent mechanism induced increased visceral pain. This aberrant pain signalling resulted from histamine receptor H1-mediated sensitization of visceral afferents. Moreover, injection of food antigens (gluten, wheat, soy and milk) into the rectosigmoid mucosa of patients with irritable bowel syndrome induced local oedema and mast cell activation. Our results identify and characterize a peripheral mechanism that underlies food-induced abdominal pain, thereby creating new possibilities for the treatment of irritable bowel syndrome and related abdominal pain disorders.

Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T0 plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T1 generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T0 plants, we could remove foreign DNA easily by genetic segregation in the T1 generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.

Atmospheric cold plasma treatment of soybean protein isolate: insights into the structural, physicochemical, and allergenic characteristics.

Currently, there has been a surge of interest in revealing the interactions between plasma and food matrices. In this study, we investigated the impacts of atmospheric cold plasma (ACP) treatment on the structural, physicochemical and allergenic characteristics of soybean protein isolate (SPI). SPI dispersions were subjected to ACP treatments at different frequencies (80 to 100 Hz) and durations (1 to 10 min) to investigate the effects of exposing conditions. Results showed that ACP induced reactive oxygen species-mediated oxidation of soy proteins, resulting in modifications in the secondary and ternary structures of SPI. As a consequence, functional properties of SPI, such as emulsifying (56 to 168%, compared with control) and foaming properties (60 to 194%) were influenced by varying degrees. In addition, under certain circumstance (120 Hz, 5 min), the IgE-binding level of SPI was decreased by up to 75%, when compared to the control. Moderate treatment yielded products with improved functionality and reduced allergenicity, while extensive exposure induced a loss of vendibility due to protein aggregation. PRACTICAL APPLICATION: In this study, we demonstrated for the first time, that plasma species reacted with soybean proteins, resulting in spatial structural changes which are closely related with protein functionality and allergenicity. ACP interacts with macromolecules in aqueous systems and thus can be an alternative and promising nonthermal approach in modifying soybean proteins, whereas the exact role of different processing parameters needs to be well-elaborated.

Antigenic activity and epitope analysis of ß-conglycinin hydrolyzed by pepsin.

BACKGROUND: Soybean is among the 'big eight' allergenic foods, and ß-conglycinin, the main antigenic protein of soybean, has high levels of antigenic activity. Why the antigenic activity of soybean ß-conglycinin is not eliminated by enzymatic hydrolysis is not clear. In this study, changes in the molecular composition and antigenicity of ß-conglycinin hydrolyzed by pepsin were analyzed and it was determined whether complete sequential epitopes exist in the resulting hydrolysates. The nature and antigenic activity of protein subunits obtained after ß-conglycinin hydrolysis were also assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and competitive enzyme-linked immunosorbent assay, respectively. RESULTS: The residual antigenic activity of ß-conglycinin was 52%, α'- and α-subunits completely disappeared, the 49 kDa fraction partially disappeared, and peptides measuring 27 and 23 kDa were newly formed after 60 min of enzymatic hydrolysis. Prolonged enzymatic hydrolysis did not result in remarkable changes in these peptides; thus, the peptides show some resistance to enzymatic hydrolysis. The amino acid sequences of the peptide chains were analyzed by matrix-assisted laser desorption / ionization-time of flight mass spectrometry and aligned with the related sequences in the corresponding protein and antigen databases. Ten complete sequential epitopes were identified in the residual 49 kDa fraction, of these epitopes, two were from α-subunits and eight were from ß-subunits. CONCLUSION: The presence of complete sequential epitopes in hydrolysates obtained from the enzymatic hydrolysis of soybean is an important reason for the incomplete disappearance of the antigenic activity of ß-conglycinin. © 2020 Society of Chemical Industry.

Lactobacillus rhamnosus GG Reduces ß-conglycinin-Allergy-Induced Apoptotic Cells by Regulating Bacteroides and Bile Secretion Pathway in Intestinal Contents of BALB/c Mice.

Allergy can cause intestinal damage, including through cell apoptosis. In this study, intestinal cell apoptosis was first observed in the ß-conglycinin (ß-CG) allergy model, and the effect of Lactobacillus rhamnosus GG (LGG) on reducing apoptosis of cells in the intestine and its underlying mechanisms were further investigated. Allergic mice received oral LGG daily, and intestinal tissue apoptotic cells, gut microbiota, and metabolites were evaluated six and nine days after intervention. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis revealed that LGG intervention could reduce the incidence of cell apoptosis more effectively than natural recovery (NR). The results of 16S rRNA analysis indicated that LGG intervention led to an increase in the relative abundance of Bacteroides. Metabolite analysis of intestinal contents indicated that histamine, N-acetylhistamine, N(α)-γ-glutamylhistamine, phenylalanine, tryptophan, arachidonic acid malate, and xanthine were significantly decreased, and deoxycholic acid, lithocholic acid were significantly increased after the LGG intervention on ß-CG allergy; the decreases in histamine and N(α)-γ-glutamylhistamine were significant compared with those of NR. In conclusion, LGG reduces apoptosis of cells induced by ß-CG allergy, which may be related to regulation of Bacteroides and the bile secretion pathway.

Lactobacillus rhamnosus GG alleviates ß-conglycinin-induced allergy by regulating the T cell receptor signaling pathway.

Currently, the need for safe and effective methods for relieving allergies is an important concern. In this study, we evaluated the role of Lactobacillus rhamnosus GG (LGG) in alleviating ß-conglycinin (ß-CG)-induced allergies and elucidated the related molecular mechanisms. Typical allergy symptoms and inflammatory factors in the serum showed that LGG intervention effectively alleviated ß-CG induced allergy in mice, which was better than natural recovery (NR). Intestinal villi were restored and lower levels of CD4+ T cells infiltrated after LGG intervention. We evaluated whether LGG intervention weakened the proliferation ability of the spleen cells of allergic mice, balancing between T/B cells and Th1/Th2 and Th17/Treg cytokines. Transcriptome analysis revealed that 4106 differentially expressed mRNAs were identified by comparing the LGG group and ß-CG group, and 546 differentially expressed mRNAs were identified by comparing the LGG group and NR group. KEGG pathway analysis identified that the T cell receptor (TCR) signaling pathway was significantly enriched upon LGG intervention, and the upregulated Ifnar2 and the downregulated Tgfbr2, Il13r2 and Il4ra were further validated by qPCR analysis. Therefore, the above results fully revealed the important role of LGG in alleviating ß-CG-induced allergies.

Immunoglobulin E and immunoglobulin G cross-reactive allergens and epitopes between cow milk αS1-casein and soybean proteins.

Some infants allergic to cow milk-based formula are also sensitive to soybean-based formula. This paper aimed to explore the association of IgE and IgG cross-reactivity between αS1-casein in cow milk (CM) and soybean proteins. The IgE and IgG cross-reactive allergens and epitopes were identified using sera from infants allergic to CM or mice monoclonal antibodies. The AA sequence alignment was performed using bioinformatics software. Finally, the digestion and heating stability of the cross-reactive allergen were explored by sodium dodecyl sulfate (SDS)-PAGE and Western blotting. The results showed that the IgE and IgG cross-reactive allergen was α subunit of ß-conglycinin named Gly m Bd 60K. The IgE and IgG epitopes were the sequences at AA 319-341 and AA 164-182. No intact Gly m Bd 60K allergen could be observed after 2 min in simulated gastric fluid by SDS-PAGE. Heating did not change IgE and IgG cross-reactivity by Western blotting. Therefore, the existence of cross-reactivity between CM αS1-casein and soybean proteins possibly contributes to the frequently observed cosensitization for these allergens in cow milk-allergic patients. The same IgE- and IgG-binding epitopes of cross-reactive allergens may provide important information for elucidation of the association between IgG and IgE antibody generation.

Location of destroyed antigenic sites of Gly m Bd 60 K after three processing technologies.

Gly m Bd 60 K, which is the α subunit of ß-conglycinin, is a major soybean (Glycine max) allergen. We used high hydrostatic pressure (HHP), thermal techniques, and glycation to treat ß-conglycinin, which can effectively reduce the antigenicity of ß-conglycinin. ß-conglycinin was used to immunize New Zealand rabbits, and the antiserum had a titer > 1: 1 × 105 and an IC50 of 2.254 µg/mL. ß-conglycinin was subjected to HHP, thermal techniques, and glycation and mixed with rabbit antiserum against ß-conglycinin to obtain the site-specific antiserum. The overlapping gene fragments of Gly m Bd 60 K were amplified by polymerase chain reaction (PCR), then cloned into a T7 phage vector and packaged in vitro, the recombinant T7 phages were constructed. Indirect ELISA (iELISA) was used to locate the destroyed antigenic sites and, after three rounds of segment expression and identification, the C2-1 and C2-2 fragments were identified as destroyed antigenic sites of Gly m Bd 60 K. Allergenicity analysis showed that the C2-1 and C2-2 fragments reacted with allergic patients' serum, which indicated that the destroyed sites were allergic sites.

Identification of the linear immunodominant epitopes in the ß subunit of ß-conglycinin and preparation of epitope antibodies.

ß-conglycinin is one of the major allergens in soybean protein. The purpose of this study was to predict and to identify the major linear epitopes of the ß subunit of ß-conglycinin. Potential linear epitopes were predicted and confirmed by three immunoinformatics tools combined with the Immune Epitope Database (IEDB). Ten potential epitope peptides were synthesized by Fmoc (9-fluorenylmethoxycarbonyl) solid phase peptide synthesis and were validated by the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) using sera from soybean allergic patients. Polyclonal antibodies, which were prepared by immunizing rabbits with synthesized peptides, were used to confirm their binding ability with ß-conglycinin through western blot and dot blot assays. The results showed that 10 peptides were screened as the main epitopes for the ß subunit of ß-conglycinin. All 10 peptides (P1-P10) presented IgG binding activity, and P2 and P6 were also validated as IgE binding peptides. Moreover, the results of dot blot showed that P5 and P8 might be located inside the protein molecule. Western blot indicated that most of polyclonal antibodies were bound effectively to the ß subunit of ß-conglycinin. In addition, few polyclonal antibodies exhibited an immune cross-reaction with the α and α' subunits.