Preparation and characterization of point mutation peanut major allergen Ara h2

To express and purify the point mutation peanut major allergen Ara h 2 and preliminarily characterize the allergenicity of purified recombinant M-Ara h 2 protein,the Ara h 2 was mutated and inserted into the expression vector pET-32a(+).The vector was transformed into Escherichia coli BL21(DE3) and the protein expression was induced by IPTG.Ni2+ chelating affinity chromatography was used to purify the recombinant M-Ara h 2 protein.The allergenicity of M-Ara h 2 was examined by Western-blotting and ELISA.The ORF which contained 474 bp and encoded 157 amino acids was authenticated to be M-Ara h 2.The recombinant M-Ara h 2 protein induced by IPTG is consistent with the actual value.The affinity between recombinant M-Ara h 2 protein and IgE antibodies from pooled peanut-allergic patients serum decreased significantly compared with R-Ara h 2 identified by Western-blotting and ELISA.In this research,recombinant M-Ara h 2 protein was obtained with hypoallergenic.