加工导致β-伴大豆球蛋白致敏性降低的亚分子结构定位

The key reason of food allergy is the allergen protein contained in the soybean. The different processing methods could reduce the allergenicity of β-conglycinins, which are the major soybean allergen protein. However, the essence of structural biology of allergenicity changes has not been fully revealed. The present study is to investigate the localization of the sub-molecular structures caused by the decrease of allergenicity to β-conglycinins. Firstly, the epitopes specific antibodies to the destruction of the process will be obtained by allergy oral and antibody absorption methods. Then, the structure of β-conglycinins will be analyzed by bioinformatics method, and T7 phages will display the overlapping β-conglycinin fractions. The positive T7 phage clones will be screened and the sub molecular structures will be positioned with the epitopes specific antibodies. Finally, the key amino acid residues will be determined by amino acid replacement technology. The research can not only provide the theoretical basis for the selection of processing methods for the food industry, but also can be applied in the development of the products which can detect the desensitization effect of processed foods rapidly.

大豆所含的致敏原蛋白是引起食物过敏的首因。不同加工方法可降低大豆主要致敏原蛋白β-伴大豆球蛋白致敏性。然而，这些致敏性变化的结构生物学实质尚未得到充分揭示。本项目首先用过敏原口服、抗体吸收等方法获得加工破坏的过敏原表位特异性抗体，然后通过生物信息学方法对β-伴大豆球蛋白进行结构分析，并在T7噬菌体表面展示交错的β-伴大豆球蛋白片段，筛选与加工破坏的过敏原表位特异性抗体结合的阳性克隆，接着用同样方法精确定位其亚分子结构，最后通过氨基酸置换技术确定关键氨基酸残基。该研究不仅能为食品工业提供加工方法筛选的理论依据，也可以进一步发展出快速检测加工食品脱敏效果的应用产品。

大豆是重要的食物过敏原之一，严重影响了人们的健康。本项目利用等电点沉淀、硫酸铵分级盐析和凝胶过滤得到高纯度的β-伴大豆球蛋白，单因素试验基础上，以响应面法优化β-伴大豆球蛋白超高静压最佳处理条件为：压强455 MPa、处理时间18 min、蛋白浓度15 mg/mL，抗原抑制率为49.59%；试验确定热加工处理条件为：蛋白浓度10 mg/mL，100℃加热60 min，抗原抑制率为38.4%；糖基化处理条件为：糖质比3:1，反应温度60℃，反应时间2.5 d，抗原抑制率为42.8%。.利用提取的β-伴大豆球蛋白对新西兰兔进行免疫，制备其多克隆抗体，并利用正辛酸-硫酸铵沉淀法纯化多抗血清。经免疫学实验表明，多克隆抗体的效价为1:1.6×104，IC50 为1.58μg/mL，与大豆Gly m Bd 28K蛋白和花生蛋白的交叉反应率分别由25.28%、1.16%降至为0.79%、0.2%，与麦朊蛋白和芝麻蛋白的交叉反应率均低于0.1%。将过量的加工处理抗原分别加入到β-伴大豆球蛋白多克隆抗体，37℃温育1 h后，离心去除沉淀，得到的上清即为加工破坏抗原表位特异性抗体。.结合生物信息学对β-伴大豆球蛋白α、α’、β三个亚基进行overlapping分段，设计合成引物，通过PCR扩增亚基全长及其 overlapping片段，并连接到T7噬菌体上。包装后，得到的亚基及其overlapping重组噬菌体，经过3轮的分段表达和筛选，确定α亚基加工破坏抗原区域为C2-1（380EGQQQGEQRLQESVIVEISKEQIRALSKR408）和C2-2（404ALSKRAKSSSRKTISSEDKPFNLRSRDPIYSNKLGKFFEITPEKN448）片段，发现C2-1片段上一段线性抗原表位380EGQQQGEQRLQESVIVE396，也是致敏表位，关键氨基酸为Q382，Q383，G385，Q387；确定β亚基加工破坏抗原区域为378DNVVRQIERQVQELAFPGSAQD400，进一步证实该序列为超高压破坏抗原构象位点；确定α’亚基加工破坏抗原区域为488PHFNSKAIVVLVINEGEANIELVG511，该区域有线性位点488PHFNSKAIVVLV499且具有致敏性。

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