The aim of this work was to develop a liquid chromatography-tandem mass spectrometry method for the determination of milk allergen and egg allergen in food products.Signature peptides GGLEPINFQTAADQAR,VGINYWLAHK,VLVLD...The aim of this work was to develop a liquid chromatography-tandem mass spectrometry method for the determination of milk allergen and egg allergen in food products.Signature peptides GGLEPINFQTAADQAR,VGINYWLAHK,VLVLDTDYK,FFVAPFPEVFGK,and NAVPITPTLNR were confirmed and synthesized as the quantitative peptide of ovalbumin,α-lactalbumin,β-lactoglobulin,α_(S1)-casein andα_(S2)-casein,the relative isotope-labeled internal standards were used in the quantitative analysis.Linear range was in the range of0.5-5000.0 nmol/L for egg and milk allergen in bread,cake,cookie,rice crust and wheat flour samples with free from egg and milk,the limits of detection of milk allergens and egg allergen were in the range between0.94 mg/100 g and 56.71 mg/100 g,limits of quantification of milk allergens and egg allergen were in the range between 2.36 mg/100 g and 141.78 mg/100 g.The recoveries ranged from 76.7%to 122.8%,the relative standard deviations were in the range of 1.60%-15.60%.The developed method has been successfully used for the detection of egg and milk allergen in various food samples.展开更多
目的:克隆表达鸡蛋主要过敏原Gal d 3基因并检验其免疫活性。方法:提取鸡蛋的总RNA,采用RT- PCR方法扩增出目的基因片段,将其克隆入T载体中进行测序和分析。设计带有酶切位点的特异性引物,采用RT- PCR获得整个短的开放阅读框并将目的基...目的:克隆表达鸡蛋主要过敏原Gal d 3基因并检验其免疫活性。方法:提取鸡蛋的总RNA,采用RT- PCR方法扩增出目的基因片段,将其克隆入T载体中进行测序和分析。设计带有酶切位点的特异性引物,采用RT- PCR获得整个短的开放阅读框并将目的基因克隆到大肠杆菌表达载体pET-28a中进行诱导表达。通过Ni^(2+)亲和层析柱对重组蛋白进行纯化,采用免疫印迹(Western blotting)方法检测其IgE结合活性。结果:克隆获得了鸡蛋主要过敏原Gal d 3。基因开放阅读框为2118个碱基(包括终止密码子),编码705个氨基酸。该序列编码的蛋白等电点为6.5,相对分子质量约为76000。表达产物经亲和层析纯化,用Western blotting方法检测免疫学活性,目的蛋白具有良好的免疫原性。结论:成功地克隆和表达了鸡蛋主要变应原Gal d 3基因,该蛋白具有良好的免疫原性。展开更多
基金supported by National Key Research and Development Program of China(2019YFC1606400)Science and Technology Project of State Administration for Market Regulation(2021MK023)+1 种基金Hebei Province High-level Talent Funding Program(A201901008)Research Project of Hebei Administration for Market Regulation(2020ZD12)。
文摘The aim of this work was to develop a liquid chromatography-tandem mass spectrometry method for the determination of milk allergen and egg allergen in food products.Signature peptides GGLEPINFQTAADQAR,VGINYWLAHK,VLVLDTDYK,FFVAPFPEVFGK,and NAVPITPTLNR were confirmed and synthesized as the quantitative peptide of ovalbumin,α-lactalbumin,β-lactoglobulin,α_(S1)-casein andα_(S2)-casein,the relative isotope-labeled internal standards were used in the quantitative analysis.Linear range was in the range of0.5-5000.0 nmol/L for egg and milk allergen in bread,cake,cookie,rice crust and wheat flour samples with free from egg and milk,the limits of detection of milk allergens and egg allergen were in the range between0.94 mg/100 g and 56.71 mg/100 g,limits of quantification of milk allergens and egg allergen were in the range between 2.36 mg/100 g and 141.78 mg/100 g.The recoveries ranged from 76.7%to 122.8%,the relative standard deviations were in the range of 1.60%-15.60%.The developed method has been successfully used for the detection of egg and milk allergen in various food samples.
文摘目的:克隆表达鸡蛋主要过敏原Gal d 3基因并检验其免疫活性。方法:提取鸡蛋的总RNA,采用RT- PCR方法扩增出目的基因片段,将其克隆入T载体中进行测序和分析。设计带有酶切位点的特异性引物,采用RT- PCR获得整个短的开放阅读框并将目的基因克隆到大肠杆菌表达载体pET-28a中进行诱导表达。通过Ni^(2+)亲和层析柱对重组蛋白进行纯化,采用免疫印迹(Western blotting)方法检测其IgE结合活性。结果:克隆获得了鸡蛋主要过敏原Gal d 3。基因开放阅读框为2118个碱基(包括终止密码子),编码705个氨基酸。该序列编码的蛋白等电点为6.5,相对分子质量约为76000。表达产物经亲和层析纯化,用Western blotting方法检测免疫学活性,目的蛋白具有良好的免疫原性。结论:成功地克隆和表达了鸡蛋主要变应原Gal d 3基因,该蛋白具有良好的免疫原性。
