Identification Of Soybean ?-Conglycinin Antigen Epitope By Immune Affinity Mass Spectrometry

?-Conglycinin is one of the main antinutritional factors of soybean,and the intensity of allergic reaction is different between several species of animals.In this study,we screen and identify linear and conformational epitope of IgG from peptide which is enzymolysis from ?-Conglycinin by immunoaffinity mass spectrometry after got IgG from serum of rats,piglets and calves.This study reveals the amino acid sequence of soybean antigen epitopes,and it can guidance works of preparation monoclonal antibody and detection the content of antigen protein in feed,the differences between interspecies diversities of animals and provide logical proofs for research of animal sensitization mechanism.Test 1: purification of IgG from serum of rats,piglets and calvesThis test used the method of affinity chromatography to purification IgG from three kinds of allergic serum by affinity column,and detected IgG by ELISA.The results show that there is differences between metal-affinity the peaks which the allergic serum has been via the value of the affinity column that come from rats,piglets and calves which were sensitive by soybean.ELISA results show that there is specificity anti-?-Conglycinin IgG from serum of rats,piglets and calves in the peaks.This result show that IgG can be produced by rats,piglets,calves.This study has been acquired the antigenic epitopes of ?-Conglycinin used two methods,one method is co-immunoprecipitation after enzymolysis ?-Conglycinin.The other method is enzymolysis ?-Conglycinin co-immunoprecipitation with specific IgG which from three kinds of animal.We were commission HUADA PROTEIN company for antigen fragment of ?-Conglycinin by Q-E mass spectrometry,and compared the results with three subunits of ?-Conglycinin on NCBI(P13916,P11827 and P25974).The result shows that two methods can screen the antigenic epitopes of enzymatic ?-Conglycinin both.There are still some differences of amino acid sequence of the epitope Although there are a large number of antigenic epitopes in three animals are similar but there are a part of amino acid sequences are difference,several sequences is only belong to piglet like LFEITQR,several sequences is only belong to rat like HEWQHKQEK.The linear epitope is similar between calve,rat and piglet's linear epitope is different to others.In conclusion,there is difference between titer of calve,piglet and rat's specific IgG,and there are differences between the antigenic epitopes of ?-Conglycinin which is sensitized different kind of animals.The result can provide the theoretical basis for revealing the mechanism of the difference between different species of animals which were sensitivity by ?-Conglycinin,and provide the basis for the development of detection kit.