Molecular cloning of paramyosin, a new allergen of Anisakis simplex

Background Anisakis simplex is a fish parasite that, when accidentally inge sted by humans, may cause allergic reactions in sensitized individuals. The main objectives of our study were to: (1) construct a cDNA expression libr ary of A. simplex; (2) identify clones producing specific IgE binding prote in antigens, and (3) produce and purify the protein/s codified by the isola ted clones produced in Escherichia coli. Methods: An expression cDNA librar y from the third stage larvae (L3) of A. simplex was constructed. This libr ary was first screened with a rabbit anti A. simplex hyperimmune serum. The positive clones, identified using the rabbit serum, were rescreened with a pool of human sera containing high titers of IgE antibodies against A. sim plex. Results: Two positive clones were isolated carrying the genes which c odify for paramyosin. The paramyosin protein was produced in E. coli and pu rified. The partial sequence of a second paramyosin gene was also identifie d. The frequency of specific IgE binding to the recombinant and native form s of paramyosin using the sera of 26 A. simplex-sensitive individuals was 2 3 and 88%, respectively. Both paramyosins were able to inhibit 11% of the s pecific IgE binding to a total extract. Conclusions: We describe the primar y structure of a paramyosin of A. simplex. It can be considered as an aller gen based on its IgE binding capacity. We suggest that the recombinant prot ein does not maintain the complete allergenic properties of the native para myosin, considering its lower IgE binding capacity of the recombinant prote in. However, both proteins have the same specific IgE inhibition capacity. The recombinant protein can be produced in large quantities in E.coli. We p ropose the term Ani s 2 for this allergen. Copyright (C) 2000 S. Karger AG, Basel.