Cloning,Expression And Immunological Evaluation Of ?-gliadin From Wheat

As the main food crop,wheat is becoming more and more attention.Wheat seed storage proteins including gluten and gliadin,is the main component of wheat gluten and determines the quality of wheat gluten.Gliadin determines the dough extensibility and can be divided into four types,which are ?,?,? and ?-gliadin.While a-gliadin accounting for about 25%of total gliadin,it is the main protein causing celiac disease.In this study,pUC57-a-gliadin recombination plasmid was used for homology analysis.Target fragment was PCR with primers which didn't PCR signal peptide,then recovered target fragment and connected with express vector.The subunit of a-gliadin was expressed by E.coli and purified by 70%ethanol solution.The a-gliadin was deamidated by citric acid,then characterized the second structure of deamidated a-gliadin and its effect on the texture of noodles.Finally,bioinformatics was used to predict the epitope of ?-gliadin,the results were as follows:1.After in situ PCR,restriction endonuclease identification and sequencing identification,the target gene was successfully constructed into the expression vector pET-22b.2.The expression vector pET-22b-a-gliadin was successfully transformed into E.coli BL21(DE3),the expression of a-gliadin was induced by ImM IPTG at 37 ?,for 18 h,and purified by 70%ethanol solution.The SDS-PAGE analysis of expression products indicated that a-gliadin was successfully expressed.After deamidated by citric acid,a-helix structure decreased,?-fold structure and a-helix/?-fold ratio decreased,indicating that the molecular flexibility of deamidated a-gliadin increased.The endogenous fluorescence scanning of deamidated a-gliadin showed a red shift of?max,indicating that the internal structure of a-gliadin has been extended and the internal tryptophan exposed.Deamidated ?a-gliadin showed smooth and uniform lumps by SEM scanning,reducing the hardness and adhesion of noodles.3.The bioinformatics was used to predict the secondary structure and B cell linear epitopes of a-gliadin.These results showed that the predominant region of the a-gliadin B cell linear epitope was located or nearby amino acid 28?41.52?75?85?132?160?170?184?188 and 202?256.Phylogenetic tree analysis showed that the relationship of a-gliadin and Triticum urartu was close.