Rosmilah M, Shahnaz M, Zailatul H M Y, Noormalin A, Normilah I
Department of Biology, Faculty of Science and Mathematics, Sultan Idris Education University, 35900 Tanjong Malim, Perak, Malaysia.
Trop Biomed. 2012 Sep;29(3):467-78.
Crab is an important source of food allergen. Tropomyosin represents the main crab allergen and is responsible for IgE cross-reactivity between various species of crustaceans. Recently, other new crab allergens including arginine kinase have been identified. However, information on allergens of the local Portunidcrab is not available. Thus, the aim of this study was to identify the major allergens of Portunus pelagicus (blue swimming crab) using the allergenomics approach. Raw and cooked extracts of the crab were prepared from the crab meat. Protein profile and IgE binding pattern were demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using sera from 30 patients with crab allergy. The major allergens of the crab were then identified by two-dimensional electrophoresis (2-DE), followed by mass spectrometry analysis of the peptide digests. The SDS-PAGE of raw extract revealed approximately 20 protein fractions over a wide molecular weight range, while cooked extract demonstrated fewer protein bands. The raw extract also demonstrated a higher number of IgE reactive bands than the cooked extract. A heat-resistant protein of 36 kDa has been identified as the major allergen in both raw and cooked extracts. In addition, a heat-sensitive protein of 41 kDa was also recognized as a major allergen in raw crab. The 2-DE gel profile of the raw extract demonstrated about >100 distinct proteins spots and immunoblotting of the 2-DE profile demonstrated at least 12 different major IgE reactive spots with molecular masses between 13 to 250 kDa and isoelectric point (pI) values ranging from 4.0 to 7.0. The 36 and 41 kDa proteins were identified as the crab tropomyosin and arginine kinase, respectively by mass spectrometry. Therefore, this study confirmed that tropomyosin and arginine kinase are the major allergens of the local Portunid crab, P. pelagicus.
螃蟹是食物过敏原的重要来源。原肌球蛋白是主要的螃蟹过敏原，它导致了不同种类甲壳类动物之间的IgE交叉反应。最近，包括精氨酸激酶在内的其他新型螃蟹过敏原也已被鉴定出来。然而，关于当地梭子蟹过敏原的信息却尚未可知。因此，本研究的目的是采用过敏原组学方法鉴定远海梭子蟹（蓝蟹）的主要过敏原。从蟹肉中制备了螃蟹的生提取物和熟提取物。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）以及使用30名对螃蟹过敏患者的血清进行免疫印迹，展示了蛋白质谱和IgE结合模式。然后通过二维电泳（2-DE）鉴定螃蟹的主要过敏原，随后对肽段消化产物进行质谱分析。生提取物的SDS-PAGE显示在较宽的分子量范围内约有20个蛋白质组分，而熟提取物显示的蛋白条带较少。生提取物中IgE反应条带的数量也比熟提取物多。一种36 kDa的耐热蛋白已被鉴定为生提取物和熟提取物中的主要过敏原。此外，一种41 kDa的热敏蛋白也被认为是生螃蟹中的主要过敏原。生提取物的2-DE凝胶图谱显示约有100多个不同的蛋白质斑点，2-DE图谱的免疫印迹显示至少有12个不同的主要IgE反应斑点，分子量在13至250 kDa之间，等电点（pI）值在4.0至7.0之间。通过质谱分析，36 kDa和41 kDa的蛋白质分别被鉴定为螃蟹的原肌球蛋白和精氨酸激酶。因此，本研究证实原肌球蛋白和精氨酸激酶是当地梭子蟹——远海梭子蟹的主要过敏原。
