| Wheat was defined as one of the eight major allergenic foods by the Food and Agriculture Organization in 1995.Wheat allergy is considered to be a serious food safety issues,which belongs to a delayed anaphylaxis.There were major prevalence rate and rate of missed diagnosis,leading to a deterioration in the quality of life for wheat allergy sufferers.With China's entry into WTO,demand for food safety increases internationally.The allergic problem of wheat allergy will bring a serious challenge for flour&wheat products industry in China.Accordingly.the investigation will play an important role in preventing wheat allergy.Gliadin proteins are primary allergens in celiac disease,mainly monomeric proteins,rich in glutamine and proline,and can be classified into theα,β,γ,ω-type according to their different primary structures.The toxicity of gliadin proteins are confirmed,especiallyα-gliadin is harmful to mucous membranes.Though it's not difficult to extract gliadin,it is hard to purify a certain protein from a multiprotein complex,however,cloning and expression ofα-gliadin gene in vitro actually help to resolve this problem.The investigation was composed of three parts including molecular cloning ofα-gliadin gene and sequential analysis,expression ofα-gliadin gene in prokaryotic system,and prediction of the B cell epitopes onα-gliadin allergen.1 Wheat variety(Zhengmai 9023) was chosen as the material in this study. Wheat genomic DNA was successfully isolated from Zhengmai 9023,and then the clone vector pMD 19-T-α-gli was constructed.After the positive clones were received by antibiotics resistance gene screening and blue-white selection,the plasmids harbouringα-gliadin gene from the positive clones were confirmed using PCR in situ, restriction mapping,and DNA sequence analysis.Finally,homology search analysis proved thatα-gliadin gene was successful obtained.2 The expression vector pGEX-4T-1-α-gli were constructed,and transformed into E.coliBL21(DE3) pLysS.The positive recombinant cell was analyzed by PCR, restriction enzyme,and sequence analysis.The results showed that the construction was correct,including the size,the sequence,the position and the direction.The SDS-PAGE analysis of expression products indicated that the highest yield of GST-α-gliadin(57kD),about 33.6%of all bacterial proteins,was obtained when induction with 1.0mmol/L IPTG,7hrs.3 By using biotic softwares and network servers,the secondary structure ofα-gliadin was predicted,and the surface properties ofα-gliadin,such as hydrophilicity,flexibility,surface accessibility and antigenic index were analysed, which could be used for predicting B cell epitopes onα-gliadin.The predict results displayed that the secondary structure ofα-gliadin contained alpha-helix,beta-sheet, beta-turn and random coil structure.Based on the analysis of hydrophilicity, flexibility,accessibility,antigenicity and the secondary structure,it was shown that B cell epitopes onα-gliadin were located within or nearby amino acids 8～21,33～55, 68～113,184～194,213～235,and all the predicted epitopes containedβ-turn and random coil structure.
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