Preparation Of Polyclonal Antibody Against ?-conglycinin And Structural Charactorization Of Allergenicity Change On Beta-subunit Caused By Processing Methods

?-conglycinin is one of the main allergens of soybean,which induces an immune response to the human body,and it may induces an immune response to the human body which is danger to the health.?-conglycininis contains ?,?'and ? submits.And the research found that the ? submits also has immunological activity.According to the internal structure of antigen protein,it can be divided into linear and conformational epitope.At present,the research on epitope is mostly B cell linear epitope.Due to the complexity of protein spatial structure,there are certain limitations in prediction.Therefore,this study uses bioinformatics technology analysis method and combines the phage display technology to predict antigenicity and positioning of the molecular structure which is decreased by the ultrahigh pressure processing,It successfully identify main damaged antigen of beta subunits.By the experiment of polypeptides.It provides the basis in theory and in practice on confirm beta subunits of allergenic molecular mechanism and carrying out no allergic food,what's more,it can prevent the occurrence of food allergic diseases and provide theory and practice of allergic diseases diagnosis.The research contents are follows:A highly specific anti-?-conglycinin polyclonal antibody was prepared by immunological determination of ?-conglycinin.Anti-?-conglycinin antibody was generated in New Zealand rabbits using ?-conglycinin as antigen and purified by octanoic acid-ammonium surfate precipitation.And then taking an analysis of serum protein content,purity,titer,sensitivity and specificity before and after purification.The results showed that the serum protein got high content by experiment of SDS-PAGE.The titer and sensitivity had no obvious change by the indirect ELISA.The ratios of cross-reactivity of ?-conglycinin with Gly m Bd 28 K and peanut protein were declined from 25.28% to 0.79% and 1.16% to 0.2%.We successfully prepared the anti-?-conglycinin polyclonal antibody possessed high specificity.This study provided a basis for the detection of ?-conglycinin allergen and the identification of epitopes.The structure model of soybean major allergen beta submits is established by using the DiscoTope 2.0 server.And according to the DiscoTope 2.0 server to predict the conformational epitopes.The beta subunit of ?-conglycinin was divided into three overlapping fragments segmented(A,B and C)by bioinformatics tool.The overlapping beta subunit of ?-conglycinin genes were amplified by PCR technology and inserted into pMD-18 T vector.The recombinant plasmids was determined by PCR and double enzyme digestion,and DNA sequence analysis showed that the cloned sequence was right.The recombinant plasmid was connected with T7 phage vector after double enzyme excision,the foreign protein is expressed to Phage capsid.And the amino acid sequence was processed and destroyed by ELISA detection.This experiment through three rounds of repeated screening,finally get the amino acid sequence of 378 to 400 named �DNVVRQIERQVQELAFPGSAQ D� as the major antigen site area which is destroyed by ultrahigh pressure processing.In this experiment,a polypeptide chain was designed according to the characteristics of the amino acid sequence in the site of antigen destruction site,and the dot-blot test was carried out with polypeptide and BSA.Results show that the ?-conglycininis extracted out in laboratory is positive reaction with recombinant phage,and no reaction with synthetic peptides.Finally concluded that the amino acid sequence is the position of conformation epitope which is destroyed by pressure processing.