Localization Of Epitopes Impairing Antigenicity Of ?-conglycinin ?' Subunit

?-conglycinin is one of the proteins that cause food allergies in soybean and is a conjugated trimer containing three subunits ?,?',?,and all of three subunits have allergenicity.In the current researching reports on ?-conglycinin,there has been less research on ?-conglycinin ?' subunits.In this study,?-conglycinin ?' subunits were analyzed and predicted by bioinformatics techniques and then overlapped.The phage display proteins which have target segment were obtained by PCR,T-vector cloning and phage display technique.The ELISA reaction was performed by using rabbit antiserum prepared from ?-conglycinin,serum obtained by ultra-high pressure treatment absorbed by antigen,and antigen-absorbed serum from thermal processing treatment as primary antibody.After three rounds of overlapping segmentation,the antigenic epitopes destroyed by processing are located.Synthesis of peptides using the amino acid sequence of the targeted disrupted epitope.Detection of conjugated peptides and rabbit antiserum prepared from ?-conglycinin?ultra-high pressure treatment of antigen absorption of rabbit antiserum?heat-treated antigen absorption of rabbit antiserum by Dot-blot and ELISA.The conjugated polypeptide is capable of reacting with rabbit antiserum prepared from ?-conglycinin,and does not react with rabbit antiserum that is processed to destroy antigen absorption.It reported that the epitope may contain both linear and conformational sites.The phage display protein of this epitope can react with rabbit antiserum prepared from ?-conglycinin and react with rabbit antiserum that is processed to destroy antigen absorption,indicating that the ultra-high pressure and heat treatment methods destroied the conformational site in the epitope,but does not destroy the linear site.Finally,the antigenic epitope was confirmed to be allergenic by ELISA using the mixed serum of 12 soybean allergy patients as primary antibody.It was of great significance to study the ?-conglycinin ?' subunit,correctly guide the processing of safe foods,and prevent the occurrence of food allergic diseases.The main content of the study is as follows:1.Analyse and predict of ?-conglycinin ?' subunits using bioinformatics techniques,overlapping fragmentation and primer design based on predictions.The recombinant plasmid T-Q,T-A,T-B,T-C and phage display proteins Pt-Q,Pt-A,Pt-B and Pt-C were obtained using PCR,T-vector cloning and phage display technology.Detection by enzyme-linked immunosorbent assay revealed that the phage display protein Pt-C has the highest antigenicity,and the ultra-high pressure and heat treatment have stronger damage to Pt-C.2.Further analysis and overlapping segments of ?-conglycinin ?' subunit fragment C using bioinformatics techniques—fragments C1,C2,and C3.Using PCR,T vector cloning,phage display technology and enzyme-linked immunosorbent assay(ELISA)to construct the cloning vector and the expression,purification and identification of phage protein in the overlapping segmentation of fragment C.The conclusion is: the antigenicity of the phage-expressed protein Pt-C1 is higher than that of the other two proteins.The thermal processing treatment has a stronger destruction of Pt-C1,and the ultra-high pressure treatment strongly destroys Pt-C3.The degree of destruction of Pt-C1 antigenicity by heat treatment is higher than the destruction of Pt-C3 by ultra-high pressure.Select Pt-C1 antigen,which is more severely damaged by processing,for further identification.3.Using techniques of biological informatics,PCR,T-vector cloning,phage display,and enzyme immunoassay to analysis and overlap segmentation of ?-conglycinin ?' subunit fragment C1.Construction of the cloning vector and expression,purification and identification of phage proteins were performed on the overlapping segmentation of fragment C1.Identification of phage-expressed protein Pt-C1-Y with the highest antigenicity by ELISA.The antigenicity of Pt-C1-X is more strongly destroyed by the hyperbaric treatment,and the antigenicity of Pt-C1-Y was more strongly destroyed by the thermal processing.The degree of destruction of Pt-C1-Y antigenicity by heat treatment was higher than the destruction of Pt-C1-X by ultra-high pressure.Select Pt-C1-Y which can be better destroyed by the process for further study.The Pt-C1-Y amino acid sequence was synthesized into a polypeptide for study.4.The synthetic polypeptide(C)PHFNSKAIVVLVINEGEANIELVG,polypeptides 1,2,3 are coupled to BSA,the polypeptide was successfully coupled by SDS-PAGE.The Dot-blot method was used to find that BSA-Pep-Y,BSA-Pep-1,and BSA-Pep-2 can be recognized by polyclonal antibodies prepared from ?-conglycinin,whereas BSA-Pep-3 was not associated with ?-associated soybeans.The polyclonal antibody reaction produced by globin indicates that the fragment may have a linear binding site for ?-conglycinin.Neither BSA-Pep-Y,BSA-Pep-1,BSA-Pep-2,or BSA-Pep-3 were found to react with the processed protein to absorb serum by ELISA,further demonstrating that the fragment has linearity of ?-conglycinin.The binding site was not destroyed by processing.However,the phage display protein Pt-C1-Y can react with both the polyclonal antibody prepared from ?-conglycinin and the rabbit antiserum absorbed by the processed protein,indicating that the epitope may be a conformational epitopes containing a linear epitope.The mixed serum of 12 soybean allergy patients was used as primary antibody in ELISA to identify phage display proteins Pt-C1-Y,conjugated peptides BSA-Pep-Y,BSA-Pep-1,BSA-Pep-2,BSA-Pep Sensitization of-3: Pt-C1-Y was able to react with mixed sera from soybean allergy patients,but BSA-Pep-Y,BSA-Pep-1,BSA-Pep-2,and BSA-Pep-3 did not respond,indicating that protein Pt-C1-Y has Sensitization,whereas BSA-Pep-Y,BSA-Pep-1,BSA-Pep-2,and BSA-Pep-3 are not allergenic.