Core Epitope Analysis Of16KD Allergen From Tartary Buckwheat

Because of its balanced amino acid composition and high content of flavonoids anddietary fiber, tartary buckwheat not only widely accepted as staple worldwide, but also as afunctional food for the prevention and treatment of diabetes, hypertension, rheumatoidarthritis and other diseases in some Asian countries. However, researchers found that someallergens in tartary buckwheat can cause severe allergic reactions, which could hinder thedevelopment and utilization of tartary buckwheat products.Currently, most studies related to buckwheat allergy focused on the common buckwheat.The researchers found that6kD，9kD,16kD,19kD,22kD,24kD,60-70kD allergens in thecommon buckwheat were the potential major allergen, and researchers conducted in-depthstudies in the structure and function of those allergens. However, there are few reports on theallergens in tartary buckwheat. Only16kD,22kD,24kD and56kD allergens in tartarybuckwheat were reported. The studies of these allergens still remains in the stage of genecloning and protokaryon expression, and the analysis of their antigenic epitopes has not beenreported yet.In this study, gene sequence of16KD allergen in tartary buckwheat seeds (GenBankNO. GO496294) obtained from the cDNA library of seed-filling period, and its correspondingamino acid sequence (GenBank Accession NO. HQ829975) were served as the theoreticalbasis, and the reconstructed plasmid containing the cDNA sequence of tartary buckwheat16kD allergen pET47b-cTBW16, which has been previously constructed in our laboratory,were served as experimental material. Six B-cell epitopes and seven T-cell epitopes ofTBW16were predicted by bioinformatics softwares, and six groups of replaced amino acidsin site-directed mutagenesis were designed, which were C38G, C52G, R71N CC87GG,C100Y, K132N, respectively. The secondary and tertiary structure and functional domains ofthese six mutant proteins were simulated by SWISS-MODEL and other molecular modelingsoftwares to make sure that the introduction of mutations will not lead to significant changesin spatial conformation. Subsequently, six mutant plasmids have been properly replaced usingsite-directed mutagenesis techniques.The prokaryotic expression of six mutant proteins in E. coli BL21star (DE3) wasinduced by IPTG. The inclusion complex of six mutant proteins was refolded by Urea gradient dialysis and the purification of the six mutant proteins were conducted with thecobalt ion column chromatography and ultrafiltration separation. Finally, according to the testresults of the circular dichroism (CD), the secondary structure of TBW16and six mutantproteins nearly had no differences with the secondary structure of the original allergicproteins which confirmed the mutation did not affect the allergen spatial structure.Through ELISA experiment, the amino acids in52nd,87th,100th,132nd positions wereproved to play a key role in the allergenic reactions of tartary buchwheats as the core B-cellepitopes of TBW16. Two peptides, ENQKGRIGET (108-117) and KCGISEMECH (132-141),were verified to be the core T-cell epitopes of TBW16by peptide synthesis and dot-blottinghybridization experiments. This study laid a theoretical foundation for the the low sensitivityvaccine preparation and hypoallergenic plants.