Mapping Of IgG-Binding Epitopes On Soybean Major Allergen Gly M Bd 28k

Gly m Bd 28 K is the major allergen in soyben, and it can induce the body to produce immune response, threatening human health. IgG antibody plays an important role in immunological reaction, and can participate in two mechanisms of the four. However, there is limited imformation about how can it cause food allergy and which segment can combine with antibody, currently. Thus, we used bioinformatics tools to imitate the tertiary structure and predict the B cell epitopes of Gly m Bd 28 K in this study. Polyclonal antibodies and monoclonal antibodies against Gly m Bd 28 K were utilized to react with overlapping peptides, and three major liner IgG-binding epitopes of Gly m Bd 28 K were identified. Furthermore, three critical amino acids of one epitope were recognized by alanine scanning, and this provides data for the epitope mapping of Gly m Bd 28 K and development of hypoallergenic foods. The major contents as follows:1. Gly m Bd 28 K protein was used to immunize BALB/c mice, and the spleen of mouse numbered 2 was removed for cell fusion according to the titer and sensitivity of murine sera. Two hybridoma cell lines, 1E3 and 2F4, that secreted anti-Gly m Bd 28 K monoclonal antibodies stably were screened after three limiting dilution assays and then were injected into the mice’s abdomens to prepare monoclonal antibodies. After purification, the isotypes of the two monoclonal antibodies were all tested as IgG1. Indirect ELISA was used to detect the immunological properties of mAb1E3 and mAb2F4. The cell supernatant titers of 1E3 and 2F4 were 1:1.28×10³ and 1:6.40×10², and ascites titers were 1:4.10×10⁵ and 1:2.05×10⁵, respectively; the IC50 values of mAb1E3 and mAb2F4 to Gly m Bd 28 K were 23.99ng/mL and 95.50 ng/mL, respectively; the affinity constants were 2.62×10⁹ L/mol and 2.86×10⁹ L/mol, respectively; and the cross reaction rates of mAb1E3 with peanut protein, sesame protein, walnut protein, and Wheat glutenin were less than 0.1%.2. The model of tertiary structure of Gly m Bd 28 K was constructed by the bioinformatics tool SWISS-MODEL, basing on the tertiary structure of its homologous protein, and reliability of the model was evaluated by Deep View 4.1. DNAStar, SOPMA, BepiPred, and ABCpred were used to predict the B cell liner epitopes of Gly m Bd 28 K, and results indicated that five potential epitopes were ²⁶EGGDKKSP³³,⁹⁶RGE⁹⁸,¹⁴⁶DPST¹⁴⁹,¹⁶³GGAN¹⁶⁶, and²²²KDD²²⁴. According to the tertiary structure, we predicted six regions,³²SPKS³⁵,⁶¹GR⁶²,⁶⁴F,¹³⁵EG¹³⁶, ²⁰⁶DDSHAPSL²¹³, and ²¹⁵TKFLQLKKDDKEQQ²²⁸, that were related to the conformational epitopes of Gly m Bd 28 K by DiscoTope 2.0.3. Bioinformatics tools like DNAStar were uesd to analyze the primary and secondary structure of Gly m Bd 28 K and its structure characteristics such as hydrophilicity,flexibility and surface accessibility. Thirty-four overlapping peptides that covered the entire sequence of Gly m Bd 28 K were synthesized, and two polyclonal antibodies against Gly m Bd 28 K together with three monoclonal antibodies（mAb2D9,mAb3F5, and mAb3H6） against recombinant Gly m Bd 28 K and two（mAb1E3 and mAb2F4） against natural Gly m Bd 28 K were utilized to identify the IgG-binding regions of Gly m Bd 28 K. Three dominant peptides corresponding to ²⁸GDKKSPKSLFLMSNS⁴²（G28-S42）, ⁵⁶LKSHGGRIFYRHMHI70（L56-I70）, and ¹⁵⁴ETFQSFYIGGGANSH¹⁶⁸（E154-H168） were recognized. L56-I70 is the most important epitope, and a competitive ELISA indicated that it could inhibit the binding of mAb3F5 to Gly m Bd 28 K protein. Alanine scanning of L56-I70 documented that F64, Y65, and R66 were the critical amino acids of this epitope.