甲壳类水产品肌浆蛋白中过敏原致敏性消减的分子机理

Recently, with constantly growing in the consumption of crustaceans products in China, the incidence of seafood allergies become more frequent. Crustaceans were reported as one of the eight categories allergenic foods by the Food and Agriculture Organization. As the important allergens in myosinogen of crustaceans, arginine kinase (AK), sarcoplasmic calcium-binding protein (SCP), triosephosphate isomerase (TIM), and filamin C (FLN c) have been well studied. Moreover, it has been reported that food processing can affect the allergenicity of food allergens. However, the study on the processing effects of crustaceans allergens were very limited. Therefore, further study is necessary to investigate how the processing of crustaceans allergens contribute to their structures and immunoglobulin E (IgE)-binding epitopes. Our previous researches indicated that the important allergens in myosinogen of crustaceans AK, SCP, TIM, and FLN c possessed the similar aggregation properties, cross-linking, and co-epitopes, which are concerned with the allergenicity and cross-reactivity. However, the mechanism of protein aggregation, cross-linking, and cross-reactivity, as well as processing treatment on its allergenicity are still unclear. Thus, in this project, the relationship between the similar properties and allergenicity will be investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, circular dichroism spectroscopy, cell model and animal model etc. And the epitopes will be mapped by the method of phage display, and comparison of these allergens will be carried out to verify the structure-activity relationship of the allergens. Furthermore, a detection method based on the co-epitopes of these allergens will be established. Meanwhile, thermal processing, high pressure processing, Maillard reaction, and enzymatically cross-linking will be employed to investigate the effects of structure variation on the allergenicity of allergens. Furthermore, the potential mechanism will be illustrated based on the digestibility, IgE-binding activity of allergens, and the expression level of cytokine in sensitized animals. In a word, the effect of IgE-binding activity, antigenic epitopes, and conformation of the allergens with similar properties, as well as their protein structure on the allergenicity will be fully analyzed and confirmed. Meanwhile, the variation in food processing of the allergenicity of these allergens will also be identified. The results of this program are believed to be helpful to establish the basis for the hypoallergenic food by technology of food processing or molecule biological engineering.

随着甲壳类水产品的国民生活消费量不断提升，其过敏发病率有明显增加的趋势，但目前仍无消除其致敏性的有效方法。本课题组研究发现，甲壳类水产品肌浆蛋白中过敏原（精氨酸激酶、肌质钙结合蛋白、磷酸丙糖异构酶、细丝蛋白C）在热聚合、空间结构、抗原表位等方面具有相似特性，这些特性与致敏性及交叉反应存在一定关联，然而其关联规律、结构基础及作用机理尚不明确。基于前期研究基础，本项目拟进一步分析明确甲壳类水产品肌浆蛋白中相似过敏原及其抗原表位，创建基于表位肽抗体的相似过敏原检测方法；结合体内外多级评价体系，解析甲壳类水产相似过敏原特性与致敏性及交叉反应的关联规律，探讨相似过敏原的免疫反应机制；探究高温高压、美拉德反应、酶法交联等食品加工中甲壳类水产过敏性的变化规律，明确过敏原结构变化对致敏性的影响，深层次揭示食品加工定向改变相似过敏原致敏性的分子机理，以期为甲壳类水产食品致敏性消减及相关产业发展提供理论依据。

食物过敏作为食品安全的热点问题在全球引起了广泛关注，甲壳类水产品是联合国粮农组织公布的八大类过敏食物之一，水产品在国民生活消费和出口创汇方面所占的比重逐年增加，然而甲壳类水产品过敏发病率有明显增加的趋势，目前仍无消除其致敏性的有效方法。.本课题组完成甲壳类水产品肌浆蛋白过敏原精氨酸激酶（arginine kinase，AK）、肌质钙结合蛋白（sarcoplasmic calcium-binding protein，SCP）、磷酸丙糖异构酶（triosephosphate isomerase，TIM）和细丝蛋白C（filamin C，FLN c）的纯化鉴定及克隆表达，比较发现天然过敏原与重组过敏原的理化特性及免疫结合活性相似；通过X射线衍射获得AK、TIM及FLN c的晶体结构，利用噬菌体展示技术、一珠一化合物结合生物信息学技术明确了甲壳类水产品肌浆蛋白中过敏原抗原表位，解析了结构与表位之间的构效关系；基于抗原表位对过敏原进行分子改造，并利用动物敏化模型揭示了抗原表位对甲壳类水产品过敏原致敏性的影响；探究了高温高压、美拉德反应等食品加工中甲壳类过敏原致敏性的变化规律，明确了过敏原结构变化对致敏性的影响，深层次揭示食品加工定向改变过敏原致敏性的分子机理，并基于此研究获得了低致敏性蟹类及虾类产品；此外利用生物信息学及蛋白质组学，在分子水平及蛋白水平上说明了甲壳类过敏原存在交叉反应性。.综上，对甲壳类过敏原克隆表达、抗原表位的分析为开发低过敏性衍生物、甲壳类水产品过敏性疾病控制提供了理论基础。同时通过高温压力、美拉德反应等消减了甲壳类过敏原的致敏性，为今后生产低致敏性水产制品提供了新思路和新方法。

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