| Peanut allergy affects the health of 5% of children and 1.4~2% of adults worldwide,which is one of the major threats to human health,and Ara h 1 is the major allergen in peanut.Current studies have shown that the allergenicity of peanuts changed by the ways of heat treatment.Our previous studies have shown that the allergenicity of peanuts might be reduced by boiling and frying,while increased by roasting.It is speculated that glycation reaction during roasting affects the allergenicity of peanut allergens.In this study,the Ara h 1 advanced glycation end products(AGE-Ara h 1)was obtained by dry-heating method after recombinant Ara h 1(R-Ara h 1)was expressed in prokaryotic expression.The structural changes of Ara h 1 protein induced by dry-heating treatment were analyzed.BALB/c mice and RBL cells were used as allergenicity models to evaluate the effect of glycation on the allergenicity of Ara h 1.Moreover,the mechanism of glycation modification affecting the allergenicity of Ara h 1 was investigated,and the effects of glycation modified allergens on every stages of allergic reaction were revealed.The main research contents and conclusions are as follows: 1 Acquisition of R-Ara h 1 protein and AGE-Ara h 1.Total RNA of peanut was extracted and reverse transcribed into cDNA.Ara h 1 gene was amplified by PCR.The sequence was cloned into pET-28 a vector.The expression plasmid of Ara h 1-pET-28 a vector was constructed,verified and sequenced.The sequenced plasmid was transferred into the bacterial strain Rosetta(DE3)for protein expression.Then the recombinant protein expressed in inclusion body was dissolved and dialyzed.After purified by Ni affinity chromatography,the recombinant protein was identified as Ara h 1 protein of peanut by mass spectrometry.Further,dry-heating treatment was used to simulate glycation reaction.With the increase of treatment time,the content of free amino group decreased,the content of bound sugar increased,fructosamine and small molecules with high fluorescence level were produced,indicating that glycation reaction occurred.The advanced glycation end products characteristic structure detected by MS-MS and ELISA showed that AGE-Ara h 1 formed after dry-heating conditions(100°C,60min).2 Effect of advanced glycation modification on allergenicity of recombinant peanut Ara h 1.The allergenicity of AGE-Ara h 1 was studied by BALB/c mice and RBL cell sensitization evaluation models.BALB/c mice were subcutaneously injected with R-Ara h 1 and AGE-Ara h 1 protein.The results showed that,AGE-Ara h 1 sensitized mice had lower body weight growth rate,higher spleen mass fraction,more severe inflammatory and congestion in jejunum and lung,higher degranulation of mast cells and basophils compared with R-Ara h 1.In addition,the high levels of Th2 cytokines(IL-4,IL-5,IL-13),Ara h 1 specific antibodies(IgE,IgG),histamines in the serum of mice,TSLP gene expression in intestine and lung indicated that the AGE-Ara h 1 sensitized mice had more severe Th2 allergic reaction.RBL cell experiments showed that the serum of mice sensitized by AGE-Ara h 1 was more functional in basophil degranulation,leading to significantly high hexosaminidase(β-HXA)release.In conclusion,AGE-Ara h 1 has more allergenicity,that is,advanced glycation modification up-regulate the allergenicity of R-Ara h 1.3 The mechanism of advanced glycation modification up-regulating the allergenicity of Ara h 1.The effects of AGE-Ara h 1 at different stages of allergic reaction were analyzed by gastric juice digestion assay,IgE binding assay,dendritic cells and RBL cell model.The results showed that advanced glycation modification decreased the digestibility of Ara h-1.Dendritic cells had higher uptake of AGE-Ara h 1,and AGE-Ara h 1 could up-regulate the RAGE expression and IL-10 release.Finally,although advanced glycation modification slightly reduced the IgE binding ability of R-Ara h 1,it increased the ability to degranulate the RBL cells,leading to more severe allergic reactions. |
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