2005 Fiscal Year Final Research Report Summary
Detection and analysis of wheat allergen candidate proteins reacting to the serum of allergy patients
| Project/Area Number |
16613012
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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 |
食の安全
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| Research Institution | National Food Research Institute |
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Principal Investigator |
KITTA Kazumi National Food Research Institute, Research Planning & Coordination Division, Food Hygiene Team, Senior Researcher, 企画調整部・食品衛生対策チーム, 主任研究官 (60353926)
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| Co-Investigator(Kenkyū-buntansha) |
KAWAMOTO Shinichi National Food Research Institute, Research Planning & Coordination Division, Food Hygiene Team, Head, チーム長 (20169775)
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| Project Period (FY) |
2004 – 2005
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| Keywords | wheat / allergen candidate protein / two-dimensional electrophoresis / immunoblotting |
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| Research Abstract |
Many people, especially babies and infants, suffer from food allergies, and the prevalence of this pathology has been on the increase recently, particularly in the developed countries. Our objective in the study was to analyze allergenic proteins employing proteomics technique to cover the wide range of protein. Wheat flour of the cultivar Hokushin (Triticum aestivum) was used for the analysis of wheat protein. Isoelectric focusing (IEF) was performed in the range of pH 3 - 10. As a second-dimensional electrophoresis, SDS-polyacrylamide gel electrophoresis (PAGE) with the Tris-glycine buffer system was chosen. Although the conventional SDS-PAGE demonstrated an excellent resolving power of proteins, it did not allow an efficient separation of proteins below 15 kDa. In order to obtain a sufficient separation of proteins below 15 kDa, various kinds of electrophoresis system was compared. Electrophoresis patterns of standard protein markers were compared on various types of gel. SDS-PAGE u
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sing the Tris-Tricine buffer system and acetic acid-urea (AU) gel demonstrated an improved separation in the region of 15 kDa. However, Tris-Tricine gel used as a second-dimensional electrophoresis resulted in a background smear toward the bottom of the gel, and the heavily distorted pattern partially obscured proteins. The AU-PAGE as a second-dimensional electrophoresis allowed an efficient separation of the proteins with masses below 20 kDa. On an AU gel, the smeared background observed on Tris-Tricine gel was not observed. Therefore, the conventional SDS-PAGE and AU-PAGE were chosen for the analysis of wheat protein. Sera of radio-allergosorbent test (RAST) score positive for wheat were obtained from six patients. For the immunodetection of the allergenic proteins of wheat, the electrophoresed gel was electroblotted to polyvinylidene difluoride (PVDF) membranes and immunolabeled with these sera. The molecular weight and isoelectric point (pI) of proteins recognized by the patients' sera were calculated with Image Master 2D Elite. Among them, some proteins were subjected to the MS analysis and identified as the known proteins. Less
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