Research On The Immunodetction Of The Main Allergic Proteins In Soybean

In this work,αsubunit of soybeanβ-conglycinin was obtained with cutting gel technique, and its purity was above 93%. The polyclonal antibody PcAb-60K-A againstαsubunit was prepared using the purifiedαsubunit. Meanwhile the epitope ⁸⁵EQDERQFPFPR⁹⁵ ofαsubunit ofβ-conglycinin based on the B-cell epitopes prediction was chosen to synthesize as hapten, and then immunogen and plate-coating antigen were prepared by coupling the hapten to KLH and BSA respectively. The polyclonal antibody PcAb-60K-B and monoclonal antibody McAb-60K were produced with the immunogen. McAb-60K was identified as being of IgG1 isotype withκlight chain, and its titer could reach 67,000. PcAb-60K-A could recognizeαandα′subunit, but PcAb-60 K-B and McAb-60 K could exhibit high specificity toαsubunit and wouldn't provoke immunologic reaction withα.' andβsubunit ofβ-conglycinin and other proteins of soybean meal. A competitive enzyme-linked immunosorbent assay with high accuracy and reproducibility based on McAb-60K and the palte-coating antigen was developed to determineαsubunit, which shown an IC50 value of 4.42 ng/mL, and the linear portion of the curve was 0.65-29.84 ng/mL.A full-length cDNA sequence of soybean allergen P34 was synthesized and inserted into the prokaryotic expression vector pET-28a (+). The recombinant P34 protein was expressed in E.coli BL21(DE3), and then was purified with His-bind affinity chromatography, which purity quotient was over 92%. The polyclonal antibody PcAb-P34 against P34 with high specificity was prepared using recombinant P34 protein as antigen, whose titer was 729,000. PcAb-P34 wouldn't recognize the other proteins of soybean meal except P34. An indirect enzyme-linked immunosorbent assay based on PcAb-P34 and recombinant P34 was developed to detect P34, which R² of standard curve of the assay was 0.9831.A full-length soybean allergen Gly m Bd 28K cDNA sequence was synthesized and inserted into the prokaryotic expression vector pET-28a(+). The Gly m Bd 28K fusion protein was expression in E.coli BL21(DE3). The recombinant protein was purified with His-Bind affinity chromatography, which purity quotient is over 90%. The polyclonal antibody PcAb-28K against Gly m Bd 28K was prepared using recombinant Gly m Bd 28K, whose titer was 27,000. PcAb-28K wouldn't cause cross reactions with other proteins of soybean meal but Gly m Bd 28K. An indirect enzyme-linked immunosorbent assay was developed based on recombinant Gly m Bd 28K protein and PcAb-28K. The R² of the curve could reach 0.9910.The detection results with the immunoassays were established in this study showed that the content ofαsubunit, immunoreactive P34 and Gly m Bd 28K in soybean seeds were 46.8μg/mg, 4.62μg/mg and 2.96μg/mg respectively, and in soybean meal were 8.9μg/mg, 3.91μg/mg and 1.21μg/mg separately. The content ofα-subunit, immunoreactive P34 and Gly m Bd 28K in fermented soybean meals were significantly reduced. Saccharomyces cerevisiae was the most effective strain for desensitizationαsubunit in the research, and then followed by Cadida utilis, Endomycopsis fibuligera, Geotrichum candidum and Bacillus subtilis. The P34 desensitization efficiency of Cadida utilis and Saccharomyces cerevisiae were higher than others, and then followed by, Endomycopsis fibuligera, Geotrichum candidum and Bacillus subtilis. The immunoreactive P34 couldn't be detected when the soybean meals were fermented by Cadida utilis, Saccharomyces cerevisiae or Endomycopsis fibuligera for 72 h. The immunoreactive Gly m Bd 28K couldn't be found from the fermented soybean meals which were fermented for 72 h. Cadida utilis was the most effective strain for desensitization the immunoreactive Gly m Bd 28K, then followed by Saccharomyces cerevisiae, Endomycopsis fibuligera, Geotrichum candidum and Bacillus subtilis.In addition, the full-length A1a and A3 acidic peptide of glycinin cDNA sequence were synthesized and inserted into the prokaryotic expression vector pET-30a (+) separately. The A1a and A3 fusion protein were expressed in BL21 (DE3) in soluble form with the induction of 0.8 mmol/L IPTG. The expression levels of them were highest in 5 h induction. The recombinant proteins were purified with His-bind affinity chromatography respectively, and their purity were 90% and 91%. And western blotting analysis revealed that the recombinant proteins had immunological activity, because they could react with the serum of soybean meal allergic piglet. The results are helpful to develop immunodetection assays for detection glycinin from soybean and soybean meals.