Molecular cloning and expression of a new major allergen, ani s 14 from anisakis simplex

Abstract

The nematode Anisakis simplex is a representative parasite infecting marine animals. When third stage larvae of A. simplex infecting fish and squids are ingested by humans, individuals previously sensitized by this parasite may experience IgE-mediated allergic reactions. So far, as many as 13 kinds of proteins (Ani s 1-13) have been identified as A. simplex allergens but several more unknown allergens are suggested to exist. In this study, therefore, chemiluminescent immunoscreening of an expression cDNA library constructed from the third stage larvae was conducted to identify a new allergen. As a result, an IgE-positive clone coding for a 23.5 kDa protein (named Ani s 14) composed of 217 amino acid residues was isolated. The regions 4-147 and 34-123 of Ani s 14 share 31% identity with the region 796-940 of Ani s 7 and 32% identity with the region 2-91 of Ani s 12, respectively. Recombinant Ani s 14 was successfully expressed in Escherichia coli as a His-tagged protein and shown to be IgE reactive to 14 (54%) of 26 sera from Anisakis-allergic patients. In conclusion, Ani s 14 is a new major allergen of A. simplex that is specific to Anisakis-allergic patients.