Expression of a recombinant protein immunochemically equivalent to the major Anisakis simplex allergen Ani s 1.
BACKGROUND
Anisakis simplex is a nematode which can parasitize humans, producing anisakiasis and can induce immunoglobulin-(Ig)-E-mediated allergic symptoms. Parasite recombinant proteins, such as the major allergen Ani s 1, may be useful tools to avoid misdiagnosis of A simplex allergy due to cross-reactivity when whole parasite extracts are used.


OBJECTIVE
To obtain Ani s 1 allergen as a recombinant protein with IgE-binding properties similar to its natural counterpart.


METHODS
Ani s 1-encoding cDNA was amplified by polymerase chain reaction and cloned. The allergen was expressed in Escherichia coli as a nonfusion protein. Natural and recombinant Ani s 1 were investigated by means of Western blotting, enzyme allergosorbent test, enzyme-linked immunosorbent assay (ELISA), and ELISA inhibition using sera from 53 patients with A simplex allergy.


RESULTS
Residues of the amino acid sequence of the encoded protein were 99.4% identical to the reported one. Purified rAni s 1 was obtained with a yield of 2 mg/L of culture while the yield of the natural counterpart was only 50 micro/g of larvae. rAni s 1 reactivity was not significantly different from that of the natural allergen; the correlation was excellent (p = 0.92, P < .001). ELISA-inhibition experiments showed that the dose-response inhibition curve obtained with rAni s 1 overlapped with that of nAni s 1. In an enzyme allergosorbent analysis, 86.8% of the A simplex-allergic patient sera reacted to rAni s 1.


CONCLUSIONS
Recombinant Ani s 1 is immunochemically equivalent to its natural counterpart and therefore might be useful for the in vitro diagnosis of anisakiasis and A simplex-mediated allergy.
Ibarrola
I
I
Bial-Arístegui, R&D Department, Bilbao, Spain
Arilla
M C
MC
Herrero
M D
MD
Esteban
M I
MI
Martínez
A
A
Asturias
J A
JA
D000485:Allergens / Q000096:biosynthesis* / Q000235:genetics* / Q000276:immunology; D000818:Animals; D017129:Anisakiasis / Q000097:blood / Q000175:diagnosis* / Q000276:immunology; D017163:Anisakis; D000909:Antibodies, Helminth / Q000097:blood / Q000235:genetics / Q000276:immunology*; D000947:Antigens, Helminth / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D018509:DNA, Helminth / Q000235:genetics / Q000276:immunology; D004926:Escherichia coli; D015801:Helminth Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D006801:Humans; D006969:Hypersensitivity, Immediate / Q000175:diagnosis / Q000469:parasitology; D007120:Immunochemistry; D007073:Immunoglobulin E / Q000097:blood / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*
D000485:Allergens; D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D002135:Calcium-Binding Proteins; D018509:DNA, Helminth; D015801:Helminth Proteins; D011994:Recombinant Proteins; C412511:troponin-like protein, Anisakis simplex; D007073:Immunoglobulin E