cDNA cloning and molecular identification of the major oyster allergen from the Pacific oyster Crassostrea gigas

Patrick S Leung, K. H. Chu

Research output: Contribution to journalArticle

50 Citations (Scopus)

Abstract

Background: Shellfish is one of the most common food allergens. Despite the recent cloning and molecular identification of the major heat stable crustacean allergens in shrimp, lobster and crab, there have been no similar studies on molluscs to which a significant portion of populations allergic to shellfish are also hypersensitive. Recent biochemical evidence suggests that tropomyosin is also an allergen in molluscs, but data on the molecular cloning, nucleotide sequencing, expression and IgE binding to mollusc tropomyosin are lacking. Objective: This study was undertaken to clone, identify and determine the primary structure of a major IgE-reactive mollusc allergen in oyster at the DNA and protein level. Methods: We constructed an expression cDNA library from the Pacific oyster Crassostrea gigas. This library was screened for IgE binding clones using sera from 15 subjects with a well-documented history of type I hypersensitivity reactions to oysters. An IgE reactive clone was selected and sub-cloned into plasmids for nucleotide sequence determination and expression in E. coli. Results: We identified a 1.3-kb cDNA designated as Cra g 1.03. Expression of Cra g 1.03 in plasmid vector pGEX produced a 59-kDa recombinant fusion protein reactive to the IgE antibodies from patients with oyster allergies but not non-allergic controls. Cra g 1.03 has an open reading frame of 233 amino acids and demonstrates marked similarity in amino acid composition and peptide sequence with mollusc and crustacean tropomyosins. Absorption of oyster allergic sera with Cra g 1.03 totally removed IgE reactivity to oyster extract. Moreover, absorption of allergic sera with recombinant shrimp tropomyosin (Met e 1), lobster tropomyosin (Pan s 1) and crab tropomyosin (Cha f 1) removed most of the IgE reactivity to Cra g 1.03. Conclusion: Cra g 1.03 is the first oyster allergen identified at the molecular level. Nucleotide and amino acid comparison shows that this protein is the oyster tropomyosin.

Original languageEnglish (US)
Pages (from-to)1287-1294
Number of pages8
JournalClinical and Experimental Allergy
Volume31
Issue number8
DOIs
StatePublished - 2001

Fingerprint

Crassostrea
Ostreidae
Molecular Cloning
Tropomyosin
Allergens
Immunoglobulin E
Complementary DNA
Mollusca
Shellfish
Clone Cells
Amino Acids
Plasmids
Nucleotides
Serum
Recombinant Fusion Proteins
Immediate Hypersensitivity
Gene Library
Open Reading Frames
Libraries
Sequence Analysis

Keywords

  • cDNA
  • Cra g 1.03
  • IgE
  • Oyster allergen
  • Tropomyosin

ASJC Scopus subject areas

  • Immunology

Cite this

cDNA cloning and molecular identification of the major oyster allergen from the Pacific oyster Crassostrea gigas. / Leung, Patrick S; Chu, K. H.

In: Clinical and Experimental Allergy, Vol. 31, No. 8, 2001, p. 1287-1294.

Research output: Contribution to journalArticle

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abstract = "Background: Shellfish is one of the most common food allergens. Despite the recent cloning and molecular identification of the major heat stable crustacean allergens in shrimp, lobster and crab, there have been no similar studies on molluscs to which a significant portion of populations allergic to shellfish are also hypersensitive. Recent biochemical evidence suggests that tropomyosin is also an allergen in molluscs, but data on the molecular cloning, nucleotide sequencing, expression and IgE binding to mollusc tropomyosin are lacking. Objective: This study was undertaken to clone, identify and determine the primary structure of a major IgE-reactive mollusc allergen in oyster at the DNA and protein level. Methods: We constructed an expression cDNA library from the Pacific oyster Crassostrea gigas. This library was screened for IgE binding clones using sera from 15 subjects with a well-documented history of type I hypersensitivity reactions to oysters. An IgE reactive clone was selected and sub-cloned into plasmids for nucleotide sequence determination and expression in E. coli. Results: We identified a 1.3-kb cDNA designated as Cra g 1.03. Expression of Cra g 1.03 in plasmid vector pGEX produced a 59-kDa recombinant fusion protein reactive to the IgE antibodies from patients with oyster allergies but not non-allergic controls. Cra g 1.03 has an open reading frame of 233 amino acids and demonstrates marked similarity in amino acid composition and peptide sequence with mollusc and crustacean tropomyosins. Absorption of oyster allergic sera with Cra g 1.03 totally removed IgE reactivity to oyster extract. Moreover, absorption of allergic sera with recombinant shrimp tropomyosin (Met e 1), lobster tropomyosin (Pan s 1) and crab tropomyosin (Cha f 1) removed most of the IgE reactivity to Cra g 1.03. Conclusion: Cra g 1.03 is the first oyster allergen identified at the molecular level. Nucleotide and amino acid comparison shows that this protein is the oyster tropomyosin.",
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