Purification and molecular cloning of a major allergen from Anisakis simplex

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...

Full description

Saved in:
Bibliographic Details
Published in:Molecular and biochemical parasitology 2004-05, Vol.135 (1), p.69-75
Main Authors: Shimakura, Kuniyoshi, Miura, Hironori, Ikeda, Kaori, Ishizaki, Shoichiro, Nagashima, Yuji, Shirai, Toshihiro, Kasuya, Shiro, Shiomi, Kazuo
Format: Article
Language:English
Subjects:
Allergen
allergens
Allergens - chemistry
Allergens - genetics
Allergens - immunology
Allergens - isolation & purification
Amino Acid Sequence
amino acid sequences
animal parasitic nematodes
Animals
Anisakis - genetics
Anisakis - immunology
Anisakis - metabolism
Anisakis simplex
Antibodies, Helminth - blood
antigen-antibody reactions
Antigens, Helminth - chemistry
Antigens, Helminth - genetics
Antigens, Helminth - immunology
Antigens, Helminth - isolation & purification
Caenorhabditis elegans - genetics
Calcium-Binding Proteins - genetics
Chromatography
Cloning, Molecular
complementary DNA
DNA, Helminth - chemistry
DNA, Helminth - isolation & purification
Enzyme-Linked Immunosorbent Assay
Escherichia coli - genetics
Escherichia coli - metabolism
gene expression
Helminth Proteins - chemistry
Helminth Proteins - genetics
Helminth Proteins - immunology
Helminth Proteins - isolation & purification
Humans
hypersensitivity
Immunoblotting
immunoglobulin E
Immunoglobulin E - blood
Molecular cloning
Molecular Sequence Data
Molecular Weight
nucleotide sequences
Open Reading Frames
patients
Protein Sorting Signals
Purification
recombinant fusion proteins
Recombinant Proteins - chemistry
Recombinant Proteins - metabolism
Sequence Alignment
Sequence Analysis, DNA
sequence homology
Trypsin - metabolism
Trypsin Inhibitors - genetics
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0166-6851
1872-9428
DOI:10.1016/j.molbiopara.2004.01.007