AIM: To develop a Brown Norway (BN) rat model to determine the potential allergenicity of novel proteins in genetically modified food.METHODS: The allergenicity of different proteins were compared, including ovalbumin...AIM: To develop a Brown Norway (BN) rat model to determine the potential allergenicity of novel proteins in genetically modified food.METHODS: The allergenicity of different proteins were compared, including ovalbumin (OVA), a potent respiratory and food allergen, bovine serum albumin (BSA), a protein that is considered to have a lesser allergenic potential,and potato acid phosphatase (PAP), a non-allergenic protein when administered to BN rats via different routes of exposure (intraperitoneally or by gavage). IgG and IgE antibody responses were determined by ELISA and PCA,respectively. An immunoassay kit was used to determine the plasma histamine level. In addition, possible systemic effect of allergens was investigated by monitoring blood pressure.RESULTS: OVA provoked very vigorous protein-specific IgG and IgE responses, low grade protein-specific IgG and IgE responses were elicited by BSA, while by neither route did PAP elicit anything. In either routes of exposure,plasma histamine level in BN rats sensitized with OVA was higher than that of BSA or PAP. In addition, an oral challenge with BSA and PAP did not induce any effect on blood pressure, while a temporary drop in systolic blood pressure in few animals of each routes of exposure was found by an oral challenge with OVA.CONCLUSION: BN rat model might be a useful and predictive animal model to study the potential allergenicity of novel food proteins.展开更多
Cow's milk allergy is mainly observed in infants and young children. Most allergic reactions affect the skin, followed by the gastrointestinal and respiratory systems. Conventional diagnosis is based on positive a...Cow's milk allergy is mainly observed in infants and young children. Most allergic reactions affect the skin, followed by the gastrointestinal and respiratory systems. Conventional diagnosis is based on positive allergy studies and evaluation of parameters including IgE and IgG1 levels, acute allergic skin response and anaphylactic shock reactions. We developed a cell membrane chromatographic(CMC)method based on human mast cells(HMC-1) for screening potential allergens in infant formula milk powders(IFMP). HMC-1 cell membranes were extracted and mixed with silica to prepare cell membrane chromatography columns(10 mm ? 2 mm i.d., 5 mm). Under the conditions of 0.2 mL/min flow rate and214 nm detection wavelength, human breast milk showed no retention. However, IFMP showed clear retention. The retained fractions were collected and analyzed through matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS). Four major milk proteins, i.e., α-casein, β-casein, α-lactalbumin, and β-lactoglobulin A, were identified. Furthermore, these proteins and β-lactoglobulin B showed clear retention on HMC-1/CMC columns. To test the degranulation effects of the five proteins, histamine and β-hexosaminidase release assays were carried out. All five proteins induced HMC-1 cells to release histamine and β-hexosaminidase. Also, we established a reversed phase liquid chromatographic(RPLC) method for the determination of the five proteins in IFMP and the results showed that 90% proteins in IFMP were α-casein and β-casein. We concluded that cow's milk proteins may be potential allergens and caseins cause more β-casein allergic risk than other proteins. This conclusion was consistent with other studies.展开更多
Objective To characterize the relationship between the refolding process of recombinant bovine β-1actoglobulin and its immunoreactivity for clinical purposes. To establish a spectral method which examine the extent o...Objective To characterize the relationship between the refolding process of recombinant bovine β-1actoglobulin and its immunoreactivity for clinical purposes. To establish a spectral method which examine the extent of recombinant allergen renaturation. Methods The refolding process of recombinant bovine β-1actoglobulin was investigated by using circular dichroism, fluorescence and synchronous fluorescence spectra. IgE-binding capacity of recombinant protein was analyzed by ELISA. In addition, bioinformatic methods were used to explain the spectral characteristics and analyze the relationship between the conformational changes and the immunoreactivity of the protein during renaturation in vitro. Results Renaturation of recombinant bovine β-1actoglobulin resulted in a more compact structure resembling the natural counterpart with stronger IgE-binding capacity. Conclusion The degree of protein renaturation Results from this study may be of help for food future. correlated with the IgE-binding capacity of the protein. allergy therapy and development of vaccination in the展开更多
随着分子生物学和基因组、蛋白组学技术的高速发展,越来越多的过敏原蛋白得到鉴定。为了提高对过敏原的研究及其在食品安全上的应用,很多机构都建立了不同类型的过敏原数据库。本文详细介绍了由美国健康与环境科学研究所(Health and Env...随着分子生物学和基因组、蛋白组学技术的高速发展,越来越多的过敏原蛋白得到鉴定。为了提高对过敏原的研究及其在食品安全上的应用,很多机构都建立了不同类型的过敏原数据库。本文详细介绍了由美国健康与环境科学研究所(Health and Environmental Sciences Institute,HESI)协调组织国际合作团队建立的一个过敏原综合数据库COMPARE (COMprehensive Protein Allergen REsource),该数据库通过高通量序列分选算法结合专家人工审核方法,从各个公共蛋白质数据库、其他过敏原数据库以及相关文献中筛选过敏原,每年更新一次。此外还开发了基于FASTA算法的序列比对工具COMPASS (COMPare Analysis of Sequences with Software),允许用户进行实时序列比对。该数据库已广泛应用于新过敏原蛋白的鉴定以及蛋白质潜在致敏性的评价,对于食品安全管理和保障有重要参考价值。本文通过对COMPARE数据库的全面介绍,旨在提高该数据库在我国的应用价值,推动过敏原分子生物信息学的研究以及食品安全的发展。展开更多
采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、双抗夹心酶联免疫吸附(enzyme-linked immunosorbent assay,ELISA)等方法,以Jug r 1含量为指标,对云南7个不同产地的泡核桃(Juglans sigillata)进行筛选,进一步利用硫酸铵分级沉淀、凝胶过滤层...采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、双抗夹心酶联免疫吸附(enzyme-linked immunosorbent assay,ELISA)等方法,以Jug r 1含量为指标,对云南7个不同产地的泡核桃(Juglans sigillata)进行筛选,进一步利用硫酸铵分级沉淀、凝胶过滤层析、液相色谱-串联质谱等手段制备Jug r 1并进行优化和鉴定,通过圆二色谱对Jug r 1的结构进行表征,最后采用双抗夹心ELISA法测定其含量。结果表明:保山隆阳产地核桃的总蛋白含量和Jug r 1含量均最高,用于后续实验;经优化得到Jug r 1的最佳硫酸铵分级沉淀分离区间为40%~80%,进一步对其进行凝胶过滤层析的最佳条件为上样质量浓度30 mg/mL、上样体积4 mL、洗脱流速1 mL/min;在此条件下纯化得到Jug r 1含量达19.90 mg/120 mg上样量,蛋白得率为16.58%;质谱分析表明该蛋白符合Jug r 1的典型特征;圆二色谱显示Jug r 1的二级结构以α-螺旋为主,多种构象共存;经“两步法”分离纯化后可获得蛋白纯度占总蛋白96%以上的Jug r 1。本结果可为深入研究Jug r 1提供科学基础,并为其他坚果致敏蛋白的分离纯化提供参考。展开更多
大豆是我国重要的粮食作物之一,其蛋白质含量高达35%~40%(m/m)。与此同时,大豆蛋白是人们日常生活中最常见的一类食物过敏原,大豆过敏已经成为了急需解决的公共安全问题。β-伴大豆球蛋白(β-Conglycinin,7S)、大豆球蛋白(Glycinin,11S)...大豆是我国重要的粮食作物之一,其蛋白质含量高达35%~40%(m/m)。与此同时,大豆蛋白是人们日常生活中最常见的一类食物过敏原,大豆过敏已经成为了急需解决的公共安全问题。β-伴大豆球蛋白(β-Conglycinin,7S)、大豆球蛋白(Glycinin,11S)、Gly m Bd 28K和Gly m Bd 30K(P34)被认为是大豆过敏原中引发机体发生过敏反应的主要成分。迄今为止,国内外对于大豆过敏尚无根治办法,唯一的预防策略是严格避免摄入来防止过敏反应的发生。但研究指出,通过特殊的加工方法或技术手段可以降低大豆过敏原的致敏性,其中以热加工法、超高压法、酶处理法和基因工程法等方法为代表的消减技术得到广泛关注。因此,该文综述了大豆过敏原的类型,常用的过敏蛋白致敏性消减技术及各项技术的优缺点,以期为低敏性大豆食品的开发提供参考。展开更多
从芝麻中提取总RNA,用反转录聚合酶链式反应得到芝麻蛋白Ses i 3基因,构建pET-22b(+)质粒表达载体,转入BL21(DE3)感受态细胞宿主表达菌中诱导表达,经镍离子亲和层析柱获得纯品目的蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示目的...从芝麻中提取总RNA,用反转录聚合酶链式反应得到芝麻蛋白Ses i 3基因,构建pET-22b(+)质粒表达载体,转入BL21(DE3)感受态细胞宿主表达菌中诱导表达,经镍离子亲和层析柱获得纯品目的蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示目的蛋白分子质量约为66 kDa。进而采用BALB/c小鼠过敏模型评价重组Ses i 3蛋白与天然Ses i 3蛋白诱发小鼠芝麻过敏反应的差别,通过测定相关过敏性指标(特异性抗体羊抗鼠免疫球蛋白(immunoglobulin,Ig)E、IgG1和IgG2a;细胞因子白细胞介素(interleukin,IL)-4、IL-5、干扰素-γ和组胺)发现,重组蛋白Ses i 3诱发小鼠芝麻过敏的能力与天然Ses i 3蛋白相似。因此,本研究通过原核表达系统获得重组的芝麻重组蛋白Ses i 3,并证实其与天然Ses i 3蛋白具有相似的免疫活性,可以用于后续的芝麻致敏性研究。展开更多
基金Supported by the State 863 Projects, No. 2001AA212291 and 2002AA212041 and the State 973 Project, No. 001CB109007
文摘AIM: To develop a Brown Norway (BN) rat model to determine the potential allergenicity of novel proteins in genetically modified food.METHODS: The allergenicity of different proteins were compared, including ovalbumin (OVA), a potent respiratory and food allergen, bovine serum albumin (BSA), a protein that is considered to have a lesser allergenic potential,and potato acid phosphatase (PAP), a non-allergenic protein when administered to BN rats via different routes of exposure (intraperitoneally or by gavage). IgG and IgE antibody responses were determined by ELISA and PCA,respectively. An immunoassay kit was used to determine the plasma histamine level. In addition, possible systemic effect of allergens was investigated by monitoring blood pressure.RESULTS: OVA provoked very vigorous protein-specific IgG and IgE responses, low grade protein-specific IgG and IgE responses were elicited by BSA, while by neither route did PAP elicit anything. In either routes of exposure,plasma histamine level in BN rats sensitized with OVA was higher than that of BSA or PAP. In addition, an oral challenge with BSA and PAP did not induce any effect on blood pressure, while a temporary drop in systolic blood pressure in few animals of each routes of exposure was found by an oral challenge with OVA.CONCLUSION: BN rat model might be a useful and predictive animal model to study the potential allergenicity of novel food proteins.
基金supported by the National Natural Science Foundation of China (No: 81230079, 81102414, 81227802)the Natural Science Basic Research Plan in Shaanxi Province of China (Program No. 2017JQ8024)
文摘Cow's milk allergy is mainly observed in infants and young children. Most allergic reactions affect the skin, followed by the gastrointestinal and respiratory systems. Conventional diagnosis is based on positive allergy studies and evaluation of parameters including IgE and IgG1 levels, acute allergic skin response and anaphylactic shock reactions. We developed a cell membrane chromatographic(CMC)method based on human mast cells(HMC-1) for screening potential allergens in infant formula milk powders(IFMP). HMC-1 cell membranes were extracted and mixed with silica to prepare cell membrane chromatography columns(10 mm ? 2 mm i.d., 5 mm). Under the conditions of 0.2 mL/min flow rate and214 nm detection wavelength, human breast milk showed no retention. However, IFMP showed clear retention. The retained fractions were collected and analyzed through matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS). Four major milk proteins, i.e., α-casein, β-casein, α-lactalbumin, and β-lactoglobulin A, were identified. Furthermore, these proteins and β-lactoglobulin B showed clear retention on HMC-1/CMC columns. To test the degranulation effects of the five proteins, histamine and β-hexosaminidase release assays were carried out. All five proteins induced HMC-1 cells to release histamine and β-hexosaminidase. Also, we established a reversed phase liquid chromatographic(RPLC) method for the determination of the five proteins in IFMP and the results showed that 90% proteins in IFMP were α-casein and β-casein. We concluded that cow's milk proteins may be potential allergens and caseins cause more β-casein allergic risk than other proteins. This conclusion was consistent with other studies.
基金supported by the Natural Science Foundation of China (30871752)the High-tech Industrialization Funding of Guangdong Province (2009B011300010)
文摘Objective To characterize the relationship between the refolding process of recombinant bovine β-1actoglobulin and its immunoreactivity for clinical purposes. To establish a spectral method which examine the extent of recombinant allergen renaturation. Methods The refolding process of recombinant bovine β-1actoglobulin was investigated by using circular dichroism, fluorescence and synchronous fluorescence spectra. IgE-binding capacity of recombinant protein was analyzed by ELISA. In addition, bioinformatic methods were used to explain the spectral characteristics and analyze the relationship between the conformational changes and the immunoreactivity of the protein during renaturation in vitro. Results Renaturation of recombinant bovine β-1actoglobulin resulted in a more compact structure resembling the natural counterpart with stronger IgE-binding capacity. Conclusion The degree of protein renaturation Results from this study may be of help for food future. correlated with the IgE-binding capacity of the protein. allergy therapy and development of vaccination in the
文摘随着分子生物学和基因组、蛋白组学技术的高速发展,越来越多的过敏原蛋白得到鉴定。为了提高对过敏原的研究及其在食品安全上的应用,很多机构都建立了不同类型的过敏原数据库。本文详细介绍了由美国健康与环境科学研究所(Health and Environmental Sciences Institute,HESI)协调组织国际合作团队建立的一个过敏原综合数据库COMPARE (COMprehensive Protein Allergen REsource),该数据库通过高通量序列分选算法结合专家人工审核方法,从各个公共蛋白质数据库、其他过敏原数据库以及相关文献中筛选过敏原,每年更新一次。此外还开发了基于FASTA算法的序列比对工具COMPASS (COMPare Analysis of Sequences with Software),允许用户进行实时序列比对。该数据库已广泛应用于新过敏原蛋白的鉴定以及蛋白质潜在致敏性的评价,对于食品安全管理和保障有重要参考价值。本文通过对COMPARE数据库的全面介绍,旨在提高该数据库在我国的应用价值,推动过敏原分子生物信息学的研究以及食品安全的发展。
文摘采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、双抗夹心酶联免疫吸附(enzyme-linked immunosorbent assay,ELISA)等方法,以Jug r 1含量为指标,对云南7个不同产地的泡核桃(Juglans sigillata)进行筛选,进一步利用硫酸铵分级沉淀、凝胶过滤层析、液相色谱-串联质谱等手段制备Jug r 1并进行优化和鉴定,通过圆二色谱对Jug r 1的结构进行表征,最后采用双抗夹心ELISA法测定其含量。结果表明:保山隆阳产地核桃的总蛋白含量和Jug r 1含量均最高,用于后续实验;经优化得到Jug r 1的最佳硫酸铵分级沉淀分离区间为40%~80%,进一步对其进行凝胶过滤层析的最佳条件为上样质量浓度30 mg/mL、上样体积4 mL、洗脱流速1 mL/min;在此条件下纯化得到Jug r 1含量达19.90 mg/120 mg上样量,蛋白得率为16.58%;质谱分析表明该蛋白符合Jug r 1的典型特征;圆二色谱显示Jug r 1的二级结构以α-螺旋为主,多种构象共存;经“两步法”分离纯化后可获得蛋白纯度占总蛋白96%以上的Jug r 1。本结果可为深入研究Jug r 1提供科学基础,并为其他坚果致敏蛋白的分离纯化提供参考。
文摘大豆是我国重要的粮食作物之一,其蛋白质含量高达35%~40%(m/m)。与此同时,大豆蛋白是人们日常生活中最常见的一类食物过敏原,大豆过敏已经成为了急需解决的公共安全问题。β-伴大豆球蛋白(β-Conglycinin,7S)、大豆球蛋白(Glycinin,11S)、Gly m Bd 28K和Gly m Bd 30K(P34)被认为是大豆过敏原中引发机体发生过敏反应的主要成分。迄今为止,国内外对于大豆过敏尚无根治办法,唯一的预防策略是严格避免摄入来防止过敏反应的发生。但研究指出,通过特殊的加工方法或技术手段可以降低大豆过敏原的致敏性,其中以热加工法、超高压法、酶处理法和基因工程法等方法为代表的消减技术得到广泛关注。因此,该文综述了大豆过敏原的类型,常用的过敏蛋白致敏性消减技术及各项技术的优缺点,以期为低敏性大豆食品的开发提供参考。
文摘从芝麻中提取总RNA,用反转录聚合酶链式反应得到芝麻蛋白Ses i 3基因,构建pET-22b(+)质粒表达载体,转入BL21(DE3)感受态细胞宿主表达菌中诱导表达,经镍离子亲和层析柱获得纯品目的蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示目的蛋白分子质量约为66 kDa。进而采用BALB/c小鼠过敏模型评价重组Ses i 3蛋白与天然Ses i 3蛋白诱发小鼠芝麻过敏反应的差别,通过测定相关过敏性指标(特异性抗体羊抗鼠免疫球蛋白(immunoglobulin,Ig)E、IgG1和IgG2a;细胞因子白细胞介素(interleukin,IL)-4、IL-5、干扰素-γ和组胺)发现,重组蛋白Ses i 3诱发小鼠芝麻过敏的能力与天然Ses i 3蛋白相似。因此,本研究通过原核表达系统获得重组的芝麻重组蛋白Ses i 3,并证实其与天然Ses i 3蛋白具有相似的免疫活性,可以用于后续的芝麻致敏性研究。
