花生蛋白Ara h1分子结构变化及其对致敏活性的影响

Peanut protein is an ideal resource for food processing, however it is also an important food allergen seriously threatening human health, with the highest food allergy lethality rate. This project takes Ara h1, the major peanut allergen in China, as the research object, and treats it with diverse modification processing approaches, including heat treatment, hyper-pressure, and glycosylation. Regulatory mechanisms of modification processing on sensitization of peanut protein Ara h1 will be explored based on measuring the changes of protein compositions and conformations, sensitizations of in vivo and in vitro mastocytes degranulation processes, and the structure-function relationship between the above two factors. On the level of molecular structure, protein conformation changes before and after modifications will be analyzed by circular dichroism and NMR, molecular compositions of the allergic peptides will be analyzed by simulation of gastrointestinal digestion. On the cellular level, changes of medium secretion will be detected by ELISA, changes of cell proliferation capacities will be checked by the methods of isotope- incorporation and CKK-8 method, gene expression changes on transcription level will be calculated by RFFQ- PCR. On the animal level, organization form and pathological changes will be observed by electron microscope. In summary, desensitization mechanisms of diverse approaches will be systematically disclosed on in vitro, in vivo, and molecular levels, so as to provide theoretical basis for the development of desensitivition methods for peanuts in future.

花生蛋白是一种理想的食品加工原料，同时也是一种致死率较高的食物过敏原，严重危害人类健康。本课题以花生蛋白中主要的过敏原Ara h1为研究对象，采用热处理、超高压及糖基化等不同改性加工方法对其进行处理，研究改性前后蛋白分子组成及构象的变化、体内体外肥大细胞脱颗粒过程致敏性的变化以及两者之间的构效关系，揭示改性对花生蛋白Ara h1致敏性的影响机制。在分子结构上，拟利用圆二色光谱和核磁共振分析改性前后蛋白结构的变化，模拟胃肠消化分析致敏肽段分子组成；在细胞层面上，利用ELISA法考察各介质分泌性的变化，同位素掺入法和CKK-8法检测细胞增殖能力变化，实时荧光定量PCR法检测转录水平基因表达量变化；在动物层面上，采用电镜等方法观察组织形态和病理学变化。从而在体内、体外以及分子水平综合揭示各种改性加工方法对花生Ara h1致敏性的影响规律，为今后开发安全有效的脱敏方法奠定理论基础。

花生致敏在世界范围内的发病率较高，在中国较低，这可能是由于中国居民通常对花生进行热加工（烘烤、水煮、油炸）后加以食用。本项目对花生致敏原Ara h1进行提取纯化及重组表达，并对热加工过程中，花生主要过敏原的结构及致敏性变化进行研究，以探明致敏结构变化与致敏性变动之间的构效关系。具体结果包括：.（1）对于原花生中Ara h1的纯化：浸提缓冲液选择PBS（含0.9% NaCl，pH 7.4），硫酸铵沉淀的百分比为100%，阴离子交换色谱柱选择DEAE-Sephrase Fast Flow，洗脱缓冲液选择含盐量为0.4 mol/L的PBS缓冲液。对于重组Ara h 1的制备，结果表明300 mM IPTG（isopropyl-β-D-thiogalactopyranoside）22 ℃诱导菌液22 h时蛋白表达量最高；15 mM十二烷基磺酸钠的添加可有效将蛋白释放在上清液中，使用含50 mM、100 mM咪唑洗脱液分别洗脱2次、1次后可得到纯度较高且免疫原性良好的重组Ara h 1。.（2）对热处理后rAra h 1蛋白的结构及致敏性进行分析。结果表明：水煮可以降低rAra h 1的致敏性，但致敏性降低和加热时间长度之间没有线性关系。rAra h 1在加热前期倾向于降解，而当加热时长达到20分钟时，小分子片段可聚集为粒径较大的聚集体。保守的二级结构如α-螺旋随着加热时间的延长表现含量降低，表明热加工对蛋白的二级结构造成了破坏。加热过程中的结构解离与重排会导致致敏表位的缺失和破坏，这可能是造成rAra h 1致敏性降低的直接原因。.（3）在花生致敏原的消化过程中，胃蛋白酶在2 min内就开始水解高分子量蛋白质，并使分子量低于10 kDa的多肽大量富集。Ara h 1和Ara h 3迅速水解，但仍有抗酶解片段，Ara h 2/6对胃蛋白酶具有抗性。经过120 min消化后，烘烤、水煮和原花生中可溶性组分比10 min消化后样品的IgE结合能力低，且水煮花生降低的最多。消化后的rAra h 1均可通过NF-kB通路刺激小肠细胞Caco-2 产生炎症因子，进而促发下游免疫反应，这可能是热加工后的致敏原仍然保持其致敏性的原因。