Preparation And Allergencity Assessment Of Cashew Allergen Ana O 3 And Its Mutants

Cashew allergy was a common tree nut allergy,and the incidence of cashew allergy was on the rise with the cashew consumption increasing.Ana o 3,one of the main allergenic proteins in cashew,could be recognized by 81% of cashew allergy patients’ serum.The sensitization of cashew allergen was closely related to its allergenic epitope.Actually,hypoallergenic Ana o 3 could be produced by recombinant expression of the allergen mutants which the main epitope gene was deleted.Therefore,it was worthy to prepare and evaluate the allergencity of the allergens and their mutants.The main methods,results and conclusions of this work were presentd as follows:1.The cashew allergen Ana o 3 was prepared by using raw cashews step by step follow the technique of crushing and defatting,low-salt extraction of crude protein,freeze-drying,anion exchange chromatography and ultrafiltration concentration.The prepared Ana o 3 was identified by electrophoresis,liquid chromatography-tandem mass spectrometry and immunoblotting.It was established a rapid and efficient purification method of cashew allergen Ana o 3.In this method,the p H value of the phosphate buffer used is 6.8,the elution flow rate is 2 m L/min,and the sodium chloride concentration during elution is 0.1-0.3 M.The purity of the prepared Ana o 3 protein was higher than 95% identified by SDS-PAGE,and the protein extraction yield was25.36%.2.By comparing and analyzing the recombinant protein of cashew allergen Ana o3 in Escherichia coli and Pichia pastoris,it was determined that a suitable expression system was selected for the expression of Ana o 3 mutants.In this study,the p ET-32 aAna o 3 plasmid was transformed into Escherichia coli BL21,and the expression of Ana o 3 was induced by IPTG.The gene of Ana o 3 was amplified by PCR,cloned into the p Pic9 k vector,and then introduced into Escherichia coli DH5α for identification.The sequenced plasmid was electroporated into Pichia pastoris GS115 to express Ana o 3.It was shown that both Escherichia coli and Pichia pastoris could express Ana o3.However,due to the long fermentation cycle,high cost and low yield of Pichia pastoris,Escherichia coli expression system were selected for the production of the three Ana o 3 mutants.3.According to the epitope information of Ana o 3 reported previously,three peptides of AA33-44(SGREQSCQRQFE),AA57-68(KQEVQRGGRYNQ)and AA121-135(PRICSISPSQGCQFQ)were selected as the deletion fragments of the mutants to synthesize plasmids.Afterwards,the mutant plasmid was transformed into Escherichia coli BL21,and the expression of Ana o 3 mutant was induced by IPTG,followed by purification by nickel ion affinity.4.The Ig E binding ability of the the natural,recombinant Ana o 3 and its mutants was tested by immunoblotting.The potential allergencity was assessed by detecting the release levels of histamine,interleukin(IL-6,IL-8)and β-hexosaminidase(β-HEX)in the KU812 cell degranulation model.It was shown that there was no significant difference in the immune activity of the natural and recombinant Ana o 3,and the allergenicity of the three mutants was significantly decreased,where the mutant 2(the deleted fragment was AA KQEVQRGGRYNQ)had the lowest immune activity.The released level of histamine,β-HEX,IL-8 and IL-6 from the induced ku812 cells by the mutant 2 was 12.40 ng/m L,1303.89 pg/m L,293.56 pg/m L and 375.34 pg/m L,respectively.In conclusion,it was established a method for highly efficient preparing of cashew allergen Ana o 3 and soluble expression of recombinant Ana o3 mutants.The prepared cashew allergen Ana o 3 can be used as the standard material candidate,and the recombinant Ana o 3 mutant can be used as the candidate of hypoallergenic Ana o 3.