RESUMEN
BACKGROUND: Japanese hop is an important cause of weed pollinosis in East Asia. Its pollen is abundant in autumn. This pollen is known to be the cause of many allergic diseases. However, molecular characteristics of its allergens have not been elucidated. OBJECTIVE: In this study, we produced recombinant proteins of allergen homologues from Japanese hop by the analysis of expressed sequence tags (EST), and evaluated its allergenicity. METHODS: cDNA library was constructed using as little as 50 ng of total RNA from Japanese hop pollen. Allergen homologues were identified by the initial screening of 963 EST clones. Recombinant proteins were overexpressed in the E. coli expression system and purified using Ni-nitrilotriacetic acid-agarose. Purified proteins were analyzed by ELISA. RESULTS AND DISCUSSION: Japanese hop pathogenesis-related 1 protein (PR-1) shares 37.0 to 44.4% of amino acid sequence identity with Art v 2, Cuc m 3, and Cyn d 24. Pectin methyl esterase (PME) shows 23.2 to 50.2% of identities to Act d 7, Ole e 11, and Sal k 1. Polygalacturonase (PGs) shows 16.7 to 19.3% of identities to Phl p 13, Cry j 2, Cha o 2, Jun a 2, Pla a 2, and Pla or 2. IgE antibodies from Japanese hop allergy patients' sera recognized PR-1 (3.4%), PME (13.8%), PGs (3.7%), and profilin (13.8%), respectively. CONCLUSION: Novel allergenic components were identified, even though low IgE reactivity was displayed reflecting the low degree of cross-reactivity with other pollen allergens. We believe that these molecules have worth further studies.
Asunto(s)
Alérgenos , Hidrolasas de Éster Carboxílico , Humulus , Proteínas de Plantas , Polen , Poligalacturonasa , Alérgenos/química , Alérgenos/genética , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/genética , Humanos , Humulus/química , Humulus/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Polen/química , Polen/genética , Poligalacturonasa/química , Poligalacturonasa/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
BACKGROUND/AIMS: Accurate diagnosis and the effects of allergen-specific immunotherapy for pollinosis are greatly dependent on the potency and stability of the extract. This study aimed to examine factors, such as temperature and storage buffer composition, that affect the stability of allergen extracts from pollens of allergenic importance in Korea. METHODS: We prepared four pollen allergen extracts from ragweed, mugwort, Japanese hop, and sawtooth oak, which are the most important causes of seasonal rhinitis in Korea. Changes of protein and major allergen concentration were measured over 1 year by Bradford assay, two-site enzyme-linked immunosorbent assay, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reconstitution of the lyophilized allergen extract in various buffers and stored at room temperature (RT, 18°C to 26°C) or refrigerated (4°C). RESULTS: More than 90% of the original protein concentration in all four extracts examined was detected over 1 year when 50% glycerol was added and refrigerated, whereas 57.9% to 94.5% remained in the extracts at RT. The addition of 50% glycerol to the storage buffer was found to prevent protein degradation at RT. Amb a 1, a major allergen of ragweed, was almost completely degraded in 9 weeks at RT when reconstituted in a buffer without 50% glycerol. However, 55.6% to 92.8% of Amb a 1 content was detected after 1 year of incubation at 4°C in all buffer conditions except 0.3% phenol. CONCLUSION: Addition of 50% glycerol as well as refrigeration was found to be important in increasing the shelf-life of allergen extracts from pollens of allergenic importance.