还原协同加热处理对花生过敏原Ara h2结构及抗原性的影响

花生过敏能引起荨麻疹、呕吐甚至休克等症状,通常是终生性过敏。本研究运用加热处理、半胱氨酸还原对Ara h2进行单独和复合处理,采用圆二色谱、紫外扫描光谱、间接竞争酶联免疫吸附法分别检测蛋白加工前后的结构与抗原性变化。结果显示,单一的加热和半胱氨酸还原处理过的蛋白结构与抗原性只发生部分变化。而复合处理中,在加热条件下,半胱氨酸还原蛋白的能力明显增强,还原后的蛋白二级结构与三级结构发生大幅变化,抗原性显著降低。研究结果表明,二硫键对于蛋白Ara h2结构稳定具有重要作用,二硫键被还原和加热过程破坏后,蛋白的抗原性和结构即发生重大变化。还原协同加热处理有望大幅改善2S球蛋白的结构和致敏性,可以作为蛋白脱敏加工的备选方法。     

A potential practical approach to reduce Ara h 6 allergenicity by gamma irradiation

Peanut allergen Ara h 6 was isolated and irradiated at 1, 3, 5, or 10 kGy, and a whole peanut protein extract (WPPE) was also treated by irradiation. Alteration in structure of Ara h 6 was characterised by circular dichroism (CD) spectroscopy, ultraviolet (UV) absorption spectroscopy, fluorescence spectroscopy and SDS–PAGE, and antigenicity was evaluated by immunoblotting and indirect ELISA with anti-Ara h 6 polyclonal antibody. Irradiation induced significant changes in the secondary and tertiary structures of Ara h 6, and the antigenicity of both purified Ara h 6 and WPPE were reduced upon increasing the irradiation doses. Moreover, a good correlation between the loss in α-helix and IgG binding to Ara h 6 was observed. This indicated that irradiation might be an efficient approach to reduce or eliminate peanut allergenicity.     

脂质过氧化物对花生过敏原致敏性的影响

目的 阐明加工过程中脂质过氧化物对花生过敏蛋白Ara h 1结构和过敏原性的影响.方法 通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳技术,圆二色谱法和内源荧光光谱法研究不同脂质过氧化物[2,2-偶氮二(2-甲基丙基咪)二盐酸盐(2,2'-azobis(2-methylpropanimidamide)dihydrochlorid...     

花生过敏原Ara h 2.02 二级结构和B 细胞抗原表位预测

为预测花生过敏原Ara h 2.02 的二级结构和B 细胞抗原表位,通过Genbank 数据库搜索到Ara h 2.02 基因,并推导出其相应的氨基酸序列,采用SOPMA 和DNAStar 软件预测该蛋白质的二级结构以及通过Jameson-Wolf 法、Kyte-Doolittle 法、Emini 法和Karplus-Schulz 法分别预测其抗原指数、亲水性、表面可性、柔韧性等参数,并综合分析预测该蛋白的B 细胞抗原表位。结果表明：在序列28～31、56～73、76～90、92、148、156～158、168～169 区域最可能是Ara h 2.02 蛋白的B 细胞抗原表位的优势区域。该结果将为寻找Ara h 2.02 的最佳优势表位提供支持,同时为该蛋白单克隆抗体的制备等研究提供理论依据。     

Peanut varieties with reduced Ara h 1 content indicating no reduced allergenicity

Peanut allergy is a major cause of food‐induced severe anaphylactic reactions. To date, no medical care is available to prevent and treat peanut allergy and therefore hypoallergenic peanut varieties are of considerable health political and economic interest. Major allergens that induce IgE‐responses in peanut‐sensitive patients are Ara h 1, Ara h 2 and Ara h 3/4. In order to identify hypoallergenic peanuts, commercially locally available peanut varieties were screened for their allergen content. Ara h 1‐deficient peanuts from Southeast Asia were identified by SDS‐PAGE, immunoblotting, inhibition assays and ELISA. 2‐D PAGE analyses demonstrated the different compositions of the tested extracts and revealed a number of variations of the allergen patterns of peanuts from different varieties. Mediator release experiments of these peanut extracts demonstrated similar allergenicities as compared with standard peanut extract. These results indicate that the allergenicity of peanuts with reduced Ara h 1 content might be compensated by the other allergens, and thus do not necessarily cause a reduction of allergenicity.     

超高效液相色谱-电喷雾质谱法检测花生 过敏原Ara h 2

目的建立烘培食品中花生过敏原Ara h 2的液相质谱联用定量检测方法。方法选择花生蛋白中的致敏蛋白Ara h 2作为目标蛋白,筛选出该致敏蛋白的特异肽,人工合成特异肽标准品和特异肽内标,从而建立直接检测花生致敏蛋白Ara h 2的准确定量方法。同时还对全国不同地区的20种花生中致敏蛋白Ara h 2的含量进行检测分析,初步统计得出致敏蛋白Ara h 2和花生蛋白的换算系数,并以Ara h 2作为生物标记物检测10种烘培食品中花生蛋白的残留量。结果花生样品中致敏蛋白Ara h 2的定量限为4.45μg/g,回收率在106.0%~107.8%之间。烘培食品中,定量限可达到6.23μg/g,回收率在107.0%~113.2%之间。结论本方法特异性强、灵敏度高、定量准确,具有良好的应用前景。     

离子交换层析法分离花生过敏原Ara h2的研究

为了制备出花生中重要过敏原Arah2,以生花生为材料,采用脱脂、离心、膜透析、离子交换层析等方法,纯化花生过敏原Arah2。结果显示,采用阴离子交换层析方法,制取的Arah2蛋白纯度达90%,得率为21.9%,该方法为过敏原Arah2的分离研究提供了可行的实验参数。     

花生主要过敏原Ara h 1线性B细胞表位的预测及鉴定

花生过敏由于其高致敏率和致敏严重性而引起了人们的广泛关注。Ara h 1是花生中的主要过敏原,属于Cupin超家族,通过抗原表位识别结合免疫球蛋白E引发花生过敏。本研究采用生物信息学分析花生主要过敏原Cupin超家族Ara h 1线性B细胞表位氨基酸组成,及其与Ara h 1二级、三级结构之间关系,通过质谱分析Ara h 1氨基酸序列中的抗消化肽段,并分析抗消化肽段与预测线性B细胞表位的关系。结果表明,Ara h 1的线性B细胞表位富含亲水性氨基酸和带电氨基酸;其二级结构没有明显的分布规律,具有一定的回转折叠结构;分析Ara h 1三级结构发现,表位主要位于单体之间的疏水相互作用区域,部分表位埋入三聚体构象内部;Ara h 1抗消化序列与表位之间存在部分重叠。综上,质谱检测体外模拟胃肠道消化肽段并结合表位生物信息学分析可以作为鉴定Cupin超家族线性B细胞表位的新方法。     

阴离子交换层析法分离纯化花生主要过敏原Ara h1的研究

Ara h1蛋白是花生的主要过敏原蛋白。本研究从天然的花生提取花生蛋白质,通过硫酸铵分级沉淀及阴离子交换层析法纯化花生主要过敏原Ara h1。通过聚丙烯酰胺凝胶电泳分析蛋白纯度,结合免疫印迹实验对Ara h1免疫活性进行鉴定。结果表明：采用阴离子交换层析法纯化出的Ara h1纯度达到90%以上。得率为23.1%。免疫印迹实验表明,此方法纯化出来的Ara h1仍具有免疫原性,能跟花生过敏病人血清特异结合。     

Enzymatic treatment of peanut butter to reduce the concentration of major peanut allergens

This study investigated the effects of enzymatic treatment of peanut butter on two‐major peanut allergens (Ara h 1 and Ara h 2). Home‐made and commercial peanut butter samples were treated with alpha‐chymotrypsin, trypsin or the combination of these enzymes and incubated at room temperature for 24 h or at 37 °C for 3 h. Treated peanut butter samples were sampled weekly for evaluation of total soluble proteins and extractable Ara h 1/Ara h 2. Data show that 1:1 alpha‐chymotrypsin: trypsin at 0.04% of enzyme‐to‐peanut butter ratio resulted in near complete reduction of extractable Ara h 1 and Ara h 2 respectively. Treatment of peanut butter with a combination of trypsin and alpha‐chymotrypsin resulted in a decrease in IgE‐binding, suggesting that enzymatic treatment has the potential to reduce the allergenicity. However, clinical tests are needed to confirm any reduction in allergenic potential. The amount of water used to disperse enzyme did not have significant effect on allergen reduction but affected the consistency and colour of treated products, especially when the amount of water added was above 5% of peanut butter weight.     

A two-site monoclonal antibody immunochromatography assay for rapid detection of peanut allergen Ara h1 in Chinese imported and exported foods

Food allergen labeling has not yet been implemented in China. Therefore, a gold immunochromatography assay (GICA) was developed using two monoclonal antibodies (mAb) against the peanut allergen Ara h1. The GICA was specific for standard peanut samples with a sensitivity of 10 ng/ml. Peanut protein traces extracted from 124 food products imported and exported by China Customs were easily and rapidly detected by GICA. 68 food samples originally labeled as containing peanuts were positive for Ara h1 and 54 food samples labeled as not containing peanuts were negative for Ara h1, indicating that the labels from the manufacturers were accurate. However, 2 food samples labeled as not containing peanuts tested positive for Ara h1. The present GICA provides a fast, simple, semi-quantitative method for the determination of peanut allergens in foods. This detection system can be used to ensure the safety of food imported and exported by China Customs.     

Peanut allergen (Ara h 1) detection in foods containing chocolate

Inadvertent exposure to peanut in foods poses health risks for peanut-allergic individuals that can be reduced by improving detection systems for allergen contaminants in food products and manufacturing processes. Detection of peanut in chocolate has been especially difficult. We report the optimization of conditions for measuring a major peanut allergen, Ara h 1, in chocolate with the use of a two-site monoclonal antibody sandwich enzyme-linked immunosorbent assay. Ara h 1 was extracted from peanut in the presence or absence of chocolate with phosphate buffer, salt, and three dried milks (goat, soy, or nonfat) (0 to 25% wt/vol) for 15 min at 60 degrees C or for 2.5 h at room temperature. The best conditions for Ara h 1 extraction in the presence of chocolate were 5% nonfat dry milk for 2.5 h at room temperature. Spiking experiments of chocolate with peanut confirmed improvement of the extraction: Ara h 1 was detected in extractions of 0.16 to 0.33% peanut in chocolate. Interestingly, the best conditions for Ara h 1 extraction were different for peanut alone than with chocolate, regarding time, temperature, and percentage of nonfat dry milk in the extraction buffer. In chocolate with peanut foods, the total Ara h 1 values were 10-fold higher than when products were extracted with phosphate buffer alone and could be up to 400-fold higher for individual foods. The dramatic improvement of Ara h 1 extraction should allow specific allergen monitoring in chocolate-containing food products and assessment of Ara h 1 exposure.     

Boiling peanut Ara h 1 results in the formation of aggregates with reduced allergenicity

Scope : Roasting rather than boiling and Maillard modifications may modulate peanut allergenicity. We investigated how these factors affect the allergenic properties of a major peanut allergen, Ara h 1. Methods and results : Ara h 1 was purified from either raw (N‐Ara h 1) or roasted (R‐Ara h 1) peanuts. Boiling (100°C 15 min; H‐Ara h 1) resulted in a partial loss of Ara h 1 secondary structure and formation of rod‐like branched aggregates with reduced IgE‐binding capacity and impaired ability to induce mediator release. Glycated Ara h 1 (G‐Ara h 1) formed by boiling in the presence of glucose behaved similarly. However, H‐ and G‐Ara h1 retained the T‐cell reactivity of N‐Ara h 1. R‐Ara h 1 was denatured, comprised compact, globular aggregates, and showed no evidence of glycation but retained the IgE‐binding capacity of the native protein. Conclusion : Ara h 1 aggregates formed by boiling were morphologically distinct from those formed by roasting and had lower allergenic activity. Glycation had no additional effect on Ara h 1 allergenicity compared with heating alone. Taken together with published data on the loss of Ara h 2/6 from boiled peanuts, this supports the hypothesis that boiling reduces the allergenicity of peanuts.     

花生主要过敏原Ara h1的纯化

采用硫酸铵沉淀及凝胶过滤层析方法,纯化花生主要过敏原蛋白Ara h1,通过聚丙烯酰胺凝胶电泳(SDS-PAGE)结合免疫印迹实验(Western-Blotting)进行鉴定。结果表明：可纯化出纯度90%以上的Ara h1过敏原蛋白。     

Effect of cold plasma treatment on the antigenicity of peanut allergen Ara h 1

The objective of this study was to investigate the effects of cold atmospheric plasma on the antigenicity of protein Ara h 1. Dry, defatted peanut flour (DPF), whole peanut (WP) were subjected to cold atmospheric plasma at voltage of 80 kV for different treatment durations (0, 15, 30, 45 and 60 min). The allergen samples were analyzed using SDS-PAGE, immunoblot and competitive ELISA. Furthermore, the secondary structure was examined using circular dichroism. SDS-PAGE results revealed no change in the protein intensity bands corresponding to Ara h 1 for both DPF and WP. Competitive ELISA of samples showed a reduction in antigenicity up to 43% for DPF and 9.3% for WP. Circular dichroism studies revealed modifications in secondary structure induced by plasma reactive species.Industrial relevanceCold plasma has emerged as a novel processing technique. This study provides evidence for reduction of antigenicity of Ara h1 in peanuts using cold plasma. This study also demonstrated the plasma-induced changes in protein structure at high treatment duration. The work described in this research is relevant to the processing of cereal grains and legumes wherein allergenicity is a major concern. This results provide the basis for possible industrial implementation.     

High pressure,thermal and pulsed electric‐field‐induced structural changes in selected food allergens

Scope: The effects of high‐pressure/temperature treatment and pulsed electric field treatment on native peanut Ara h 2, 6 and apple Mal d 3 and Mal d 1b prepared by heterologous expression were examined. Methods and results: Changes in secondary structure and aggregation state of the treated proteins were characterized by circular dichroism spectroscopy and gel‐filtration chromatography. Pulsed electric field treatment did not induce any significant changes in the structure of any of the allergens. High‐pressure/temperature at 20°C did not change the structure of the Ara h 2, 6 or Mal d 3 and resulted in only minor changes in structure of Mal d 1b. Ara h 2, 6 was stable to HPP at 80°C, whereas changes in circular dichroism spectra were observed for both apple allergens. However, these changes were attributable to aggregation and adiabatic heating during HPP. An ELISA assay of temperature treated Mal d 3 showed the antibody reactivity correlated well with the loss of structure. Conclusion: In conclusion, novel‐processing techniques had little effect on purified allergen structure. Further studies will demonstrate if these stability properties are retained in foodmatrices.     

基于Arah6的花生间接竞争ELISA检测方法的建立

建立了基于花生主要过敏原蛋白Arah6的间接竞争酶联免疫检测法,实现了对食物中花生的定量检测。利用花生过敏原Arah6纯品免疫新西兰大白兔,制得兔抗Arah6的多克隆抗体,以Arah6纯品作为包被抗原、自制抗体为一抗、辣根过氧化物酶标记的羊抗兔IgG为二抗,确定了包被抗原质量浓度为1μg/mL,一抗最佳稀释度为1:50000,酶标二抗稀释度为1:5000。此检测方法对Arah6的定量检测范围为16.5～10000ng/mL(折合成含花生的含量,约为165ng/mL～100μg/mL),抑制方程为抑制率I=21.418lgC-6.0633(C为Arah6的质量浓度,单位ng/mL),IC50为414.6ng/mL,相关系数R2=0.9989。该检测方法灵敏度高,检测范围广,适用于食品中花生现场快速检测。     

Development of an epitope-specific analytical tool for the major peanut allergen Ara h 2 using a high-density multiple-antigenic peptide strategy

Using the major peanut allergen Ara h 2 as an example, an analytical tool enabling the determination of immunoglobulin E (IgE)-epitopes in processed food allergens was developed. We synthesized a multiple-antigenic peptide (MAP) of the IgE-reactive linear epitope 3 (amino acid positions 27-36) of Ara h 2 and raised a monospecific antiserum against this epitope to obtain a positive control for future epitope resolved diagnostics. First, a MAP of epitope 3, having a molecular mass of 7770 Da, was synthesized, purified, and its structure confirmed by liquid chromatography-mass spectrometry (electrospray ionization) (LC-MS(ESI)), matrix assisted laser desorption/ionization-time of flight (MALDI-TOF), and Edman sequencing. The MAP was then used to raise high titer antibodies in rabbits using the adjuvant Titermax and to characterize the specificity of IgE from allergenic patients sensitized to Ara h 2. The antiserum exclusively detects Ara h 2 in crude peanut extract with a titer of 10(7) by Western blot and reacts specifically with epitope 3 shown by epitope mapping for a library of solid-phase-bound synthetic 15-mer peptides covering the entire sequence of Ara h 2. Such IgE-reactive epitopes are of high analytical relevance as they could constitute the basis for epitope-specific detection systems for use in quality control in the food industry or for forensic purposes in cases of fatal reactions to otherwise undetected peanut proteins.     

DNA 提取方法对实时荧光定量 PCR 检测花生过敏原 Ara h 2 基因的比较研究

目前,食物过敏是一个世界性的公共卫生问题,其中花生过敏最为严重。基于DNA的分子生物学检测方法目前已广泛应用于花生过敏原检测,样品DNA的提取质量会显著影响检测的灵敏度及准确率。本研究比较了5种DNA提取方法,包括十六烷基三甲基溴化铵（cetyl trimethyl ammonium bromide,CTAB）法、柱式法及3种基于磁珠纯化技术的DNA快速提取法,对生花生、煮花生、炸花生、烤花生、花生酥和花生酱6种不同加工方式的花生基质样品进行DNA提取,考察了不同方法获得的DNA浓度、纯度指标,并采用实时荧光定量PCR（quantitative real-time PCR, qPCR）对花生过敏原Ara h 2基因进行了检测分析。结果表明,月桂酰肌氨酸钠（Sodium Lauroyl Sarcosine）磁珠法（简称SLS磁珠法）的适用性广、提取率高,对于6种花生基质提取的DNA均能高效检出花生过敏原Ara h 2基因;对于花生含量为0.05%～1.00%的小麦粉二元混合物,SLS磁珠法的DNA提取率总体优于CTAB法,并且能有效提取出与花生共用一条生产线的燕麦片中污染的花生DNA,证实SLS磁珠法提取的实际样品DNA能够满足花生过敏原检测目的。本研究为花生及其制品DNA提取方法提供了参考,特别是磁珠类方法,高效快速,提取质量能够保障后续基于DNA的花生过敏原分子生物学方法检测结果的准确性。     

Allergenic characteristics of a modified peanut allergen

Attempts to treat peanut allergy using traditional methods of allergen desensitization are accompanied by a high risk of anaphylaxis. The aim of this study was to determine if modifications to the IgE-binding epitopes of a major peanut allergen would result in a safer immunotherapeutic agent for the treatment of peanut-allergic patients. IgE-binding epitopes on the Ara h 2 allergen were modified, and modified Ara h 2 (mAra h 2) protein was produced. Wild-type (wAra h 2) and mAra h 2 proteins were analyzed for their ability to interact with T-cells, their ability to bind IgE, and their ability to release mediators from a passively sensitized RBL-2H3 cell line. Multiple T-cell epitopes were identified on the major peanut allergen, Ara h 2. Ara h 2 amino acid regions 11-35, 86-125, and 121-155 contained the majority of peptides that interact with T-cells from most patients. The wAra h 2 and mAra h 2 proteins stimulated proliferation of T-cells from peanut-allergic patients to similar levels. In contrast, the mAra h 2 protein exhibited greatly reduced IgE-binding capacity compared to the wild-type allergen. In addition, the modified allergen released significantly lower amounts of beta-hexosaminidase, a marker for IgE-mediated RBL-2H3 degranulation, compared to the wild-type allergen.