Molecular cloning and expression of two new allergens from Anisakis simplex

The nematode Anisakis simplex is a marine parasite that causes allergy as well as anisakiasis. Although five Anisakis allergens have already been identified, immunoblotting studies suggested that unidentified allergens still exist. In this study, an expression cDNA library constructed from A. simplex was subjected to immunoscreening using an Anisakis-allergic patient serum, and two positive clones coding for allergens (named Ani s 5 and 6) were obtained. Ani s 5 (152 amino acid residues) is homologous with nematode proteins belonging to the SXP/RAL-2 protein family and Ani s 6 (84 amino acid residues) with serine protease inhibitors from various animals. Of the 28 patient sera examined, seven and five reacted to recombinant Ani s 5 and 6 expressed in Escherichia coli, respectively. By inhibition immunoblotting experiments using the recombinant allergens as inhibitors, natural Ani s 5 could be identified as a 15-kDa protein in the crude extract of A. simplex but natural Ani s 6 could not be identified probably due to its low expression. In conclusion, Ani s 5 and 6 are new allergens of A. simplex that are specific to some Anisakis-allergic patients.