2000 Fiscal Year Final Research Report Summary
Screening of food allergy-causing factors and study on wheat allergy.
| Project/Area Number |
11680138
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
食生活
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| Research Institution | Okayama Prefectural University |
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Principal Investigator |
KIMOTO Masumi Okayama Pref.Univ.Fac.Health and Welfare Science, Professor, 保健福祉学部, 教授 (40108866)
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| Co-Investigator(Kenkyū-buntansha) |
SASAGAWA Takayo Okayama Pref.Univ.Fac.Health and Welfare Science, Lecturer, 保健福祉学部, 講師 (10254567)
OKITA Misako Okayama Pref.Univ.Fac.Health and Welfare Science, Professor, 保健福祉学部, 教授 (70079242)
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| Project Period (FY) |
1999 – 2000
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| Keywords | Wheat / Allergen / Tri a Bd 27K / glycoprotein / cDNA |
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| Research Abstract |
Of allergic patients, many patients have been suffering from allergy eliciting by ingestion of cereals. Wheat is the most important foodstuff of the cereals in view of its yield and consumption quantity in the world. In order to elucidate the allergenicity of wheat, we screened the allergens in wheat using the sera of wheat-sensitive patients with atopic dermatitis and found about 15 allergens. Among them, a 17-kDa allergic protein (Tri a Bd 17K) had been identified as α-amylase inhibitor CM 16 with an Asn-linked sugar chain. In the present study, we attempted to purify Tri a Bd 27K, a major wheat allergen, and to elucidate its properties. Furthermore, we investigated the cloning of cDNA encoding the allergen to reveal information concerning its complete primary structure.
Tri a Bd 27K was purified to near homogeneity from wheat flour by extraction with 10 % NaCl, ammonium sulfate fractionation, Q-Sepharose chromatography, chromatofocusing and phenyl-Sepharose chromatography. The purified protein showed positive response to the PAS test, indicating that the allergen is N-Asn-linked glycoprotein. The glycoprotein was shown to have α-L-fucose and α-D-mannose by the specific lectin staining. N-Terminal amino acid sequencing showed that the allergen is an unkown protein. Further, the allergen was digested with trypsin in gel and two peptides were purified by HPLC.On the basis of the N-terminal amino accid sequences of the Tri a Bd 27K and its peptides, some degenerate oligoDNAs were synthesized to use as primers of PCR.Using poly (A)^+RNA prepared from developing wheat cotyledons, RT-PCR was done to obtain a probe suitable for the cloning of cDNA.Since the PCR products were confirmed to encode the peptide fragments of the allergen, the products were used as probes in the further experiments.
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