College of Food and Biological Engineering, Xiamen Key Laboratory of Marine Functional Food, Fujian Provincial Engineering Technology Research Center of Marine Functional Food, Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine Biological Resources, Jimei University , 43 Yindou Road, Xiamen, 361021, Fujian, P.R. China.
Department of Biochemical Science and Technology, College of Life Science, National Taiwan University , No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan.
J Agric Food Chem. 2017 Aug 2;65(30):6247-6257. doi: 10.1021/acs.jafc.7b02381. Epub 2017 Jul 21.
Shellfish allergy is a prevalent, long-lasting disorder usually persisting throughout life. However, the allergen information is incomprehensive in crab. This study aimed to identify a novel allergen in crab, show its potential in diagnosis and reduce the allergenicity by food processing. A 21-kDa protein was purified from Scylla paramamosain and confirmed as sarcoplasmic calcium binding protein (SCP) by matrix-assisted laser desorption ionization-time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Total RNA was isolated from crab muscle, and a rapid amplification of cDNA was performed to obtain an ORF of 579 bp that coded for 193 amino acid residues. According to the results of circular dichroism analysis and ELISA assay, the recombinant SCP (rSCP) expressed in Escherichia coli showed similar physicochemical and immunoreactive properties to native SCP (nSCP). Additionally, the extensive cross reactivity of SCP among different species and the bidirectional IgE cross-reactivity between nSCP and rSCP were detected by iELISA. The allergenicity of rSCP was reduced via Maillard reaction or enzymatic cross-linking reaction, which was confirmed by the results of scanning electron microscopy, dot blot, and digestion assay. A straightforward and reproducible way was developed to obtain high yields of rSCP that maintains structural integrity and full IgE reactivity, which could compensate the low specific IgE-titers of most patient sera for future diagnosis. Furthermore, the Maillard reaction and enzymatic cross-linking reaction were effective approaches for the production of hypoallergenic seafood.
贝类过敏是一种常见的、持久的疾病，通常会持续一生。然而，蟹类的过敏原信息并不全面。本研究旨在从锯缘青蟹中鉴定一种新的过敏原，展示其在诊断中的潜在应用，并通过食品加工降低其致敏性。从锯缘青蟹中纯化得到一种 21kDa 的蛋白，通过基质辅助激光解吸电离飞行时间/飞行时间质谱（MALDI-TOF/TOF-MS）鉴定为肌浆钙结合蛋白（SCP）。从蟹肌肉中提取总 RNA，通过快速扩增 cDNA 获得编码 193 个氨基酸残基的 579bp 的 ORF。根据圆二色性分析和 ELISA 试验的结果，在大肠杆菌中表达的重组 SCP（rSCP）表现出与天然 SCP（nSCP）相似的理化性质和免疫反应性。此外，通过 iELISA 检测到 SCP 在不同物种之间的广泛交叉反应性，以及 nSCP 和 rSCP 之间的双向 IgE 交叉反应性。通过美拉德反应或酶交联反应降低 rSCP 的致敏性，通过扫描电子显微镜、斑点印迹和消化试验证实了这一点。建立了一种简单、可重复的方法来获得高产量的 rSCP，其结构完整，具有完整的 IgE 反应性，可以弥补大多数患者血清中特异性 IgE 滴度较低的问题，为未来的诊断提供帮助。此外，美拉德反应和酶交联反应是生产低致敏性海鲜的有效方法。
