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目的 基于高分子量谷蛋白Tri a 26建立小麦中过敏蛋白的定性定量检测方法，对基因编辑小麦和普通对照小麦样品进行过敏蛋白含量分析与比较。方法 选择小麦中的致敏蛋白Tri a 26作为目标蛋白，筛选出该致敏蛋白的特异肽段IFWGIPALLK,人工合成特异肽段，样品用100 mmol/L含4 mol/L尿素和0.1 mol/L二硫苏糖醇的Tris-HCl缓冲液(pH 8.5)匀浆提取，提取液经半胱氨酸残基烷基化后，与胰蛋白酶混匀在37℃酶解消化16 h,采用电喷雾正离子模式下多反应监测外标法定量，测定并对比基因编辑小麦和普通对照小麦中高分子量谷蛋白Tri a 26含量。结果 特异水解肽段标准溶液在10～2000 ng/mL范围内线性关系良好(r>0.999),样本中IFWGIPALLK肽段定量限为0.328 ng/g, 3个浓度加标回收率在84.8%～95.4%,相对标准偏差为2.99%～6.12%(n=6)。基因编辑小麦和普通对照小麦Tri a 26含量为1.976～2.069 mg/g。结论 该方法灵敏度和特异性高，可以有效地应用于小麦中高分子量谷蛋白的检测，研究发现基因编辑小麦中高分子量谷蛋白Tri a 26的含量不高于普通对照小麦。
Abstract：OBJECTIVE To establish a qualitative and quantitative method for detecting allergenic proteins in wheat based on the high-molecular-weight(HMW) glutenin Tri a 26, and to analyze and compare the allergenic protein content between genetically edited wheat and conventional control wheat samples.METHODS The allergenic protein Tri a 26 was selected as the target protein, and its specific peptide IFWGIPALLK was identified and artificially synthesized. Wheat samples were homogenized and extracted with 100 mmol/L Tris-HCl buffer(pH 8.5) containing 4 mol/L urea and 0.1 mol/L dithiothreitol. After alkylation of cysteine residues, the extracts were digested with trypsin at 37 ℃ for 16 h. Quantification was performed using multiple reaction monitoring in positive-ion mode via dart spray ionization, with an external standard method. The Tri a 26 content in genetically edited wheat and conventional control wheat was measured and compared.RESULTS The standard solution of the specific hydrolyzed peptide exhibited good linearity in the range of 10-2000 ng/mL(r>0.999). The limit of quantification(LOQ) for the IFWGIPALLK peptide in the sample was 0.328 ng/g. The spike recovery rates at three concentration levels ranged from 84.8% to 95.4%(n =6), with relative standard deviations(RSDs) between 2.99% and 6.12%. The Tri a 26 content in genetically edited wheat and conventional control wheat ranged from 1.976 to 2.069 mg/g.CONCLUSION This method demonstrates high sensitivity and specificity and can be effectively applied to the detection of HMW glutenin in wheat. The study found that the Tri a 26 content in genetically edited wheat was not higher than that in conventional control wheat.
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基本信息:
DOI：10.19813/j.cnki.weishengyanjiu.2026.01.019
中图分类号:O657.63;TS210.7;R155.5
引用信息:
[1]沈昕,陈曦,杨奕,等.超高效液相色谱-质谱分析基因编辑小麦高分子量谷蛋白的过敏蛋白含量[J].卫生研究,2026,55(01):110-116.DOI:10.19813/j.cnki.weishengyanjiu.2026.01.019.
基金信息:
首都卫生发展专项(No.首发2024-2G-4341); 生物育种产品食用饲用安全评价技术标准化研究及应用推广(No.2023ZD0406309)