Construction And Expression Of Peanut Allergen Ara H8and Mutants For Antigenicity Attenuated

Background：According to the reports from FAO in1995, peanut is one of the eight kindsof food allergens. Compared to other food allergens, peanut allergy displays highrates of more serious clinical symptoms, which is not easy to rehabilitate byimmunological tolerance with the increase of age. Peanut allergy therefore arrestsmuch attention in the field of public health and food safety. To establish a novelmethod for accurate diagnosis and efficient treatment of peanut allergy poses agreat significance for clinicians. This study aims to obtain hypoallergenicrecombinant mutations of peanut allergen Ara h8, using genetic engineeringtechniques through site-specific mutagenesis, and amino acid substitution of theallergen sequence. To some extent, we expect to reduce the risk of specificallergen-based immunotherapy and to maintain the bioactivity of the peanutallergen. This work may provide the material basis for further studies on themechanisms, immunotherapy and clinical diagnosis of peanut allergy. Objective:We obtain the hypoallergenic form of the peanut allergen Ara h8to safeimmunotherapy regimens. This may provide the material basis for further studieson the mechanisms, immunotherapy and clinical diagnosis of peanut allergy.Methods:1. The encoding nucleotide sequence of peanut allergen Ara h8was retrievedfrom Gen Bank (AY328088.1). We optimized the amino acid codons based on theprinciples of E. coli codon usage priority and the degeneracy of amino acid codon.The recombinant proteins were expressed in prokaryotic expression system. Afterbeing purified by affinity chromatography, these proteins were indentified bywestern blotting.2. Antigen epitopes of Ara h8amino acid sequences were analyzed anddetermined using the online software NetMHC-II and Syfpeithi.3. The amino acids with high affinity and branched chain were replaced to getthe amino acid sequences with low antigenicity.4. The sites we determined were transformed by site-directed mutagenesis.These mutations were identified by Western blotting after being expressed andpurified.Results:1. The optimized nucleotide sequence of Ara h8was obtained, and expressionvector pET44a-Ara h8was successfully constructed and the recombinant proteinwas expressed.2. Three important epitope sites in Ara h8were resolved;3. We expressed six important mutatated proteins based on the three epitopesites.Conclusion:The gene of peanut allergen Ara h8was highly expressed in E.coli after being optimized. We obtained the high purified recombinant protein Ara h8whichwas identified as the target proteins by western blotting.Three important epitope sites in Ara h8were resolved after analyzing theantigenicity of Ara h8proteins. This work may provide the material basis forfurther studies on the mechanisms, immunotherapy and clinical diagnosis ofpeanut allergy.