Abstract： Objective To clone the coding sequence of Pseudomonas aeruginosa exotoxin A (PEA) and analyze the contributions of different amino acids to its antigenicity and tertiary structure. Methods Genomic DNA extracted from Pseudomonas aeruginosa ATCC27853 was used as template, and the specific coding region of PEA was used as primer. Touchdown PCR was applied to clone the coding region of immunotoxin-related PEA fragment. The amplified fragment recovered was subcloned into the pGEM-T vector. Positive clones were validated and selected by colony PCR, followed by further confirmation by DNA sequencing and Clustal X (1.83) alignment. The antigenicity and tertiary structure of the encoded peptide was analyzed by bioinformatic softwares BIMAS and SWISS-Model. Amino acid residue of key locus was replaced so as to investigate the effect of different amino acid for tertiary structure. Results Three clones, referred to as PE117, PE215 and PE217, were acquired. Sequence and structure analysis demonstrated that few amino acid sequence disparity existed among the three clones and the sequence registered in GenBank (the accession number is P11439), and thus affecting the antigenicity in the corresponding position. However, slight difference was hardly observed in their tertiary structures. Conclusion Based on sequence analysis, antigenicity evaluation and tertiary structural modeling, some useful information is obtained for antigenicity evaluation, thus would facilitate allergen genetic improvement by amino acid replacement in PEA.