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Molecular cloning of major allergen from Cupressus arizonica pollen: Cup a 1
Author(s) -
Aceituno E.,
Del Pozo V.,
Mínguez A.,
Arrieta I.,
Cortegano I.,
Cárdaba B.,
Gallardo S.,
Rojo M.,
Palomino P.,
Lahoz C.
Publication year - 2000
Publication title -
clinical and experimental allergy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.462
H-Index - 154
eISSN - 1365-2222
pISSN - 0954-7894
DOI - 10.1046/j.1365-2222.2000.00949.x
Subject(s) - recombinant dna , biology , pectate lyase , complementary dna , molecular cloning , microbiology and biotechnology , allergen , fusion protein , antigenicity , cloning (programming) , gene , genetics , biochemistry , antigen , allergy , immunology , enzyme , pectinase , computer science , programming language
The family Cupressaceae is a relevant source of allergens that causes winter respiratory allergies. Cloning and sequencing the major antigen of Cupressus arizonica is important for a better diagnosis and treatment of sensitized patients. To obtain a full‐length complementary DNA for Cup a 1, the major allergen of C upressus arizonica pollen. It was cloned and sequenced and the recombinant protein was expressed. Messenger RNA from Cupressus arizonica pollen was obtained and the Cup a 1 sequence was established using a 3′‐RACE system and primers based on the N‐terminal amino acid sequence. Recombinant Cup a 1 was cloned in pBluescript and expressed in a glycosylated form in rabbit reticulocytes. The cDNA was subcloned in pGEX‐5X‐1 and expressed in Escherichia coli as a fusion protein with GST. Recombinant Cup a 1 is highly homologous with the major allergens of mountain cedar (Jun a 1), Japanese cypress (Cha o 1) and Japanese cedar (Cry j 1). Cup a 1 contains three potential N‐glycosylation sites that are different from those found in Jun a 1 and Cry j 1. The cloned protein contains a pectate lyase active site identical to those of Cry j 1 and Jun a 1. The IgE from patients' sera recognizes recombinant Cup a 1, and this reactivity is higher with the glycosylated protein. Cup a 1 has been cloned and sequenced. As expected, the high degree of homology with Cha o 1, Jun a 1 and Cry j 1 explains the cross‐reactivity of conifer pollens. Different IgE reactivity with the glycosylated and non‐glycosylated protein suggests the importance of carbohydrate moieties in the IgE binding site.