Identification of novel three allergens from Anisakis simplex by chemiluminescent immunoscreening of an expression cDNA library.
Anisakis simplex is a representative nematode parasitizing marine organisms, such as fish and squids, and causes not only anisakiasis but also IgE-mediated allergy. Although 10 kinds of proteins have so far been identified as A. simplex allergens, many unknown allergens are considered to still exist. In this study, a chemiluminescent immunoscreening method with higher sensitivity than the conventional method was developed and used to isolate IgE-positive clones from an expression cDNA library of A. simplex. As a result, three kinds of proteins, Ani s 11 (307 amino acid residues), Ani s 11-like protein (160 residues) and Ani s 12 (295 residues), together with three known allergens (Ani s 5, 6 and 9), were found to be IgE reactive. Furthermore, ELISA data showed that both recombinant Ani s 11 and 12 expressed in Escherichia coli are recognized by about half of Anisakis-allergic patients. Ani s 11 and Ani s 11-like protein are characterized by having six and five types of short repetitive sequences (5-16 amino acid residues), respectively. Both proteins share as high as 78% sequence identity with each other and also about 45% identity with Ani s 10, which includes two types of short repetitive sequences. On the other hand, Ani s 12 is also structurally unique in that it has five tandem repeats of a CX(13-25)CX(9)CX(7,8)CX(6) sequence, similar to Ani s 7 having 19 repeats of a CX(17-25)CX(9-22)CX(8)CX(6) sequence. The repetitive structures are assumed to be involved in the IgE-binding of the three new allergens.
Department of Food Science and Technology, Tokyo University of Marine Science and Technology, Minato-ku, Tokyo 108-8477, Japan.
Yukihiro
Y
Kobayashi
Kenichi
K
Ohsaki
Kaori
K
Ikeda
Seiko
S
Kakemoto
Shoichiro
S
Ishizaki
Kuniyoshi
K
Shimakura
Yuji
Y
Nagashima
Kazuo
K
Shiomi
D000485:Allergens / Q000276:immunology / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000818:Animals; D017129:Anisakiasis / Q000175:diagnosis* / Q000276:immunology; D017163:Anisakis / Q000276:immunology* / Q000378:metabolism; D000909:Antibodies, Helminth / Q000097:blood; D000947:Antigens, Helminth / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D018509:DNA, Helminth / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D015723:Gene Library; D015801:Helminth Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D006801:Humans; D006967:Hypersensitivity / Q000378:metabolism; D007073:Immunoglobulin E / Q000097:blood; D008163:Luminescent Measurements / Q000379:methods*; D008969:Molecular Sequence Data
D000485:Allergens; D000909:Antibodies, Helminth; D000947:Antigens, Helminth; D018509:DNA, Helminth; D015801:Helminth Proteins; D007073:Immunoglobulin E