Expression And Identification Of Rice Major Allergen RA17

Background:With a large number of genetically modified foods rushing into the Chinesemarket, potential food security issues such as allergenicity attracted more and moreattention. Scientists are hooking on how to effectively evaluate and detect foodallergen. It is the key point to set up a standard comparator for allergenicityevaluation and detection. Rice protein harbors high nutritional value and werewidely consumed in the world, and its allergenicity is the lowest among all grains.Especially in China, most people live on rice but with seldom clinical allergy reports.It is therefore tempting to think that rice allergen could become the potentialreference control for allergenicity assessment of genetically modified foods. RA17(Rice allergen17) is reported as one of the major rice allergens. It is also arepresentative of the cross-reactive allergens between members of the Gramineaefamily. RA17therefore is the best comparator for rice allergenicity evaluation.Objective:This study aims to construct a reference standard for the allergenicityevaluation of genetically modified foods through recombination expression ofallergen RA17protein. Methods:1. The codon-optimized RA17gene was synthesized by Shanghai BoshangBiotechnology Co., Ltd.2. Expression vector of RA17-pET-44a was constructed with Strep-II tagged andsubsequently transformed into E. coli Rosetta. Parameters were optimized forprotein expressionthe of RA17.3. StrepTrapHPcolumn affinity chromatography was used to purify the target proteinwhich collected by ultrasonication, and the concentration and purity of therecombinant RA17protein were assayed using BCA protein quantification andSDS-PAGE electrophoresis technology.4. The recombinant RA17protein was identified by western blotting technique andmatrix-assisted laser desorb/ionize time-of-flight mass spectrometry.Results:1. The expression vector PET-44a-RA17was successfully constructed with thecodon optimized RA17gene.2. The recombinant RA17protein expression strain goes highest under theconditions of30oC, IPTG inducer to a final concentration of1.0mmol/L, andpersistent expression of3h.3. More than95%purity of the recombinant RA17protein was achieved andidentified as16kDa.Conclusion:Major rice allergen RA17has been recombinantly expressed as a referencestandard in China, thus facilitating the allergenicity evaluation of geneticallymodified foods and providing the experimental material for further research andmonoclonal antibody production.