Abstract
A heat-stable allergen with a molecular weight of 21 k was purified from larvae of the nematode Anisakis simplex by gel filtration, anion-exchange FPLC and reverse-phase HPLC. When analyzed by immunoblotting and ELISA, seven of eight patient sera reacted to the 21 k allergen, demonstrating that this protein is a major allergen of A. simplex. A full-length cDNA encoding the 21 k allergen was cloned by a combination of 3'RACE and screening of an expression library with DIG-labeled DNA probes. The precursor of the 21 k allergen was judged to be composed of a signal peptide (23 residues) and a mature protein (171 residues). As compared to the N-terminal amino acid sequence (up to the 17th residue) of Ani s 1 previously identified as the major allergen, the 21 k allergen has only one replacement, suggesting that the 21 k allergen belongs to the same protein family of Ani s 1. Although the 21 k allergen was found to have 30-40% sequence identity with Kunitz-type trypsin inhibitor domain containing hypothetical proteins of Caenorhabditis elegans, it lacked inhibitory activity against trypsin. The 21 k allergen was successfully expressed in Escherichia coli as a GST-fusion protein showing reactivity with IgE in patient sera.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Allergens / chemistry
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Allergens / genetics*
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Allergens / immunology
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Allergens / isolation & purification*
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Amino Acid Sequence
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Animals
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Anisakis / genetics*
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Anisakis / immunology*
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Anisakis / metabolism
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Antibodies, Helminth / blood
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Antigens, Helminth / chemistry
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Antigens, Helminth / genetics*
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Antigens, Helminth / immunology
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Antigens, Helminth / isolation & purification*
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Caenorhabditis elegans / genetics
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Calcium-Binding Proteins / genetics
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Chromatography
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Cloning, Molecular
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DNA, Helminth / chemistry
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DNA, Helminth / isolation & purification
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Enzyme-Linked Immunosorbent Assay
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Escherichia coli / genetics
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Escherichia coli / metabolism
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Helminth Proteins / chemistry
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Helminth Proteins / genetics
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Helminth Proteins / immunology
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Helminth Proteins / isolation & purification
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Humans
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Immunoblotting
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Immunoglobulin E / blood
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Molecular Sequence Data
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Molecular Weight
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Open Reading Frames
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Protein Sorting Signals
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Sequence Alignment
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Sequence Analysis, DNA
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Trypsin / metabolism
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Trypsin Inhibitors / genetics
Substances
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Allergens
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Antibodies, Helminth
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Antigens, Helminth
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Calcium-Binding Proteins
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DNA, Helminth
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Helminth Proteins
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Protein Sorting Signals
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Recombinant Proteins
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Trypsin Inhibitors
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troponin-like protein, Anisakis simplex
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Immunoglobulin E
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Trypsin