Molecular Cloning of Paramyosin, a New Allergen of <i>Anisakis simplex</i>

Abstract

Background: Anisakis simplex is a fish parasite that, when accidentally ingested by humans, may cause allergic reactions in sensitized individuals. The main objectives of our study were to: (1) construct a cDNA expression library of A. simplex; (2) identify clones producing specific IgE binding protein antigens, and (3) produce and purify the protein/s codified by the isolated clones produced in Escherichia coli. Methods: An expression cDNA library from the third stage larvae (L3) of A. simplex was constructed. This library 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. simplex. Results: Two positive clones were isolated carrying the genes which codify for paramyosin. The paramyosin protein was produced in E. coli and purified. The partial sequence of a second paramyosin gene was also identified. The frequency of specific IgE binding to the recombinant and native forms of paramyosin using the sera of 26 A. simplex-sensitive individuals was 23 and 88%, respectively. Both paramyosins were able to inhibit 11% of the specific IgE binding to a total extract. Conclusions: We describe the primary structure of a paramyosin of A. simplex. It can be considered as an allergen based on its IgE binding capacity. We suggest that the recombinant protein does not maintain the complete allergenic properties of the native paramyosin, considering its lower IgE binding capacity of the recombinant protein. However, both proteins have the same specific IgE inhibition capacity. The recombinant protein can be produced in large quantities in E. coli. We propose the term Ani s 2 for this allergen.

Immunization of Rabbits with NematodeAscaris lumbricoidesAntigens Induces Antibodies Cross-Reactive to House Dust MiteDermatophagoides farinaeAntigens

“…A major allergen of another nematode, Anisakis simplex, Ani s 2, is also paramyosin. 20) Although no experimental data suggested that paramyosin is important as a cross-reactive antigen between Ascaris and HDM antigens until recently, a very recent report states that the recombinant Blo t 11 fusion protein showed IgE reactivity in 80% of allergic subjects and 46% of ascariasis subjects, 15) suggesting that a potential role of paramyosin as an important crossreactive allergen in HDMs and Al.…”

Anisakis simplex: from Obscure Infectious Worm to Inducer of Immune Hypersensitivity

“…Ani s 2 and Ani s 3 are the two somatic allergens described so far (22,23,108,212). They are paramyosin and tropomyosin proteins, respectively, and are similar to the paramyosins and tropomyosins of other species (reviewed by Weiler [279]).…”

<i>Anisakis</i> <i>simplex</i> Recombinant Allergens Increase Diagnosis Specificity Preserving High Sensitivity

“…Ani s 1 (21 kDa) [10] belongs to the serin-type endopeptidase inhibitor family since it contains a Kunitz domain. Ani s 2 (paramyosin, 100 kDa) [11] and Ani s 3 (tropomyosin, 41 kDa) [12] are muscular proteins. Ani s 4 (9 kDa) [13] and Ani s 6 (7 kDa) [14] are protease inhibitors.…”