The Effect Of Processing Treatment On The Antigenicity Of ?-conglycinin And The Location Of Damaged Antigen Epitopes Of Gly M Bd 60K

Soybean is one of the most important source of food allergens,along with cow's milk and eggs;they are recognized as the"three major allergens"of children,which seriously affect children's health.?-conglycinin is the major soybean allergen,and the?subunit of?-conglycinin,Gly m Bd 60K,is an important allergen in susceptible populations and is the earliest recognized major allergenic protein.Processing can reduce the antigenicity of?-conglycinin effectively.However,there is still no relevant literature about the damaged antigen epitopes of Gly m Bd 60K after processing technologies using a phage display technique?PDT?.Therefore,location of damaged antigen epitopes of Gly m Bd 60K after processing technologies has a significant role in the detection of molecular mechanisms that reduce the sensitization of Gly m Bd 60K,product safe foods,and prevent the occurrence of food allergies.Purified?-conglycinin was obtained by electric-point precipitation,salt precipitate and filtration chromatography.The result of Western-Blot showed that the purified?-conglycinin could specially bind to?-conglycinin antiserum,which proved that the purified?-conglycinin had immunological activity.Based on the single-factor experiments,the effects of high hydrostatic pressure?HHP?treatment pressure,HHP treatment time and?-conglycinin concentration were optimized by response surface methodology.The results showed that the optimal process conditions for reduced antigenicity of?-conglycinin were determined as follows:HHP treatment pressure was 455 MPa,HHP treatment time was 18 min,and?-conglycinin concentration was 15 mg/m L,the predicted inhibition rate of?-conglycinin antigenicity was 49.59%.The effects of HHP treatment on the structural properties of soybean?-conglycinin showed that the HHP treatment did not change the molecular weight of?-conglycinin.Free sulphydryl?SH?groups and surface hydrophobicity?H₀?of?-conglycinin were significantly increased at pressures 200⁴00 MPa and 5¹5 min,whereas these properties decreased at the treatments above 400 MPa and 15 min.The maximum fluorescence intensity was observed at 400 MPa and 15 min.The circular dichroism?CD?data analysis revealed that the amount of?-turns and unordered structure significantly increased after HHP treatment.The results showed that the heat treatment conditions were as follows:protein concentration was 10 mg/mL,heating temperature was 100?,heating time was 60 min,under this condition,the antigen inhibition rate was 38.4%.Under conditions of protein to sugar ratio 3:1,60°C,2.5 d,the antigenicity inhibition rate was 42.8%in soy protein isolate?SPI?-glucose conjugates.Bioinformatics tools were used to predict the potential conformational epitopes of Gly m Bd 60K,and six major conformational epitopes were as follows:²³⁹NQRSPQLQN²⁴⁷,³¹⁷DNNE³²⁰,³⁷⁹EEGQQQGEQ³⁸⁷,⁴¹³SRK⁴¹⁵,⁴¹⁸SSED⁴²¹ and ⁴⁹⁷EQQQEEQQEEQ⁵⁰⁷.According to the prediction results,Gly m Bd 60K gene was overlapped and the primers were designed and synthesized.The overlapping gene fragments of Gly m Bd 60K were amplified by polymerase chain reaction?PCR?and ligated with T7 phage vector,after being packaged in vitro,the recombinant T7 phage was constructed and the overlapping fragments of Gly m Bd60K were displayed on the phage surface.The purified phage were expanded and purified to obtain A,B,C,and D phage purified proteins.Western-Blot results showed that the segmented protein can react well with antiserum against?-conglycinin and exihibited immunological activity.All recombinant phages that expressed the overlapping fragments of GIy m Bd 60K reacted with the epitope-specific antibody that was processed to locate the damaged epitope.The reaction showed that the most significant destructive fragments were in the C fragment.After further overlapped,segmented,amplified by PCR,ligated with T7 phage vector,and expressed on the surface of T7 phage.Indirect competitive ELISA was used to screen the destructive allergen epitopes that further overlapped segments of selected fragments.After several rounds of segment expression and screening,C2-1 and C2-2 fragments were identified as damaged antigen epitopes of Gly m Bd 60K after using three processing technologies,at the same time,it was found that phage-expressed proteins can react with sera from soybean allergy patients,which indicated that they have sensitization.The prediction of the secondary structure of the damaged antigen epitopes revealed that the frontal structure of the C2-1fragment was irregular coil,the later was alpha helix,and the main structure of the C2-2fragment was irregular coil.Overlapping peptide technology was used to further study the damaged allergen region.A linear epitope on the C2-1 fragment was found,which amino acid sequence was³⁸⁰EGQQQGEQRLQESVIVE³⁹⁶.Moreover,the synthesized overlapping polypeptides did not react with the epitope-specific antibody,indicating that the processing did not change the primary structure of the protein.Alanine scanning of ³⁸⁰EGQQQGEQRLQESVIVE³⁹⁶documented that Q382,Q383,G385,Q387 were the critical amino acids of this epitope.