Predicted by an antigenic program, full-length tartary buckwheat allergen (TBa) is divided into six fragments: E1, E2, E12, E3, E4 and E34. Immunological assays revealed that E1 has the greatest binding activity to patients' serum IgE. Five mutants of E1 gene (L39R, L42R, L47R, V52R and L54R) were constructed using site-directed mutagenesis and each protein was expressed in Escherichia coli BL21. Following purification by Ni2+ affinity chromatography, ELISA and dot-blot were performed for wild type E1 and its mutants using sera from buckwheat allergic patients and healthy controls. Mutants L42R, L47R, and L54R had weaker IgE binding activity to patient's sera than wild-type E1 implying that Leu42, Leu47, and Leu54 might be involved in the allergic activity of TBa.