Molecular cloning of paramyosin, a new allergen of Anisakis simplex.
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.
Pérez-Pérez
J
J
CBF LETI, SA, Research Laboratories, Universidad Autónoma de Madrid, Spain.
Fernández-Caldas
E
E
Marañón
F
F
Sastre
J
J
Bernal
M L
ML
Rodríguez
J
J
Bedate
C A
CA
D000485:Allergens / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D000818:Animals; D017163:Anisakis / Q000235:genetics* / Q000378:metabolism; D000909:Antibodies, Helminth / Q000276:immunology; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005399:Fishes / Q000469:parasitology; D015723:Gene Library; D017238:Genes, Helminth; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D007073:Immunoglobulin E / Q000276:immunology; D008969:Molecular Sequence Data; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014335:Tropomyosin / Q000235:genetics* / Q000276:immunology
D000485:Allergens; D000909:Antibodies, Helminth; D015801:Helminth Proteins; D007106:Immune Sera; C416856:PARA protein, Anisakis simplex; D011994:Recombinant Proteins; D014335:Tropomyosin; D007073:Immunoglobulin E