小麦主要过敏原CM16线性B细胞表位的预测及初步鉴定

目的：探究小麦主要过敏原α-淀粉酶/胰蛋白酶抑制剂CM16的线性B细胞表位。方法：通过提取小麦蛋白粗提物,进行体外模拟胃肠道消化,发现小麦过敏原中具有消化抗性的α-淀粉酶/胰蛋白酶抑制剂CM16的片段;进一步通过生物信息学方法分析CM16的一级氨基酸序列、二级结构,并通过同源建模确定CM16的三级结构。结果：利用生物信息学法预测出CM16的4 条线性B细胞表位后,结合质谱得到的抗消化肽段信息进行比对分析,发现其中2 条肽段存在部分重合,也证明了生物信息学分析鉴定过敏原线性B细胞表位方法的可行性和应用性。结论：通过生物信息学预测小麦过敏原CM16线性B细胞表位有助于进一步认识小麦过敏原,对小麦过敏原的识别和检测具有重要的参考和借鉴作用。     

质谱法分析烘焙对花生过敏原Ara h 1潜在致敏性的影响

为研究烘焙对花生过敏原Ara h 1潜在致敏性的影响,采用高离液序列盐溶液从鲜花生和烘焙花生中提取总蛋白,通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析烘焙前后蛋白条带变化情况,并对其中的花生主要过敏蛋白Ara h 1条带进行质谱和Swiss-Model模型分析。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示,烘焙花生蛋白出现了大分子聚合物条带以及较多弥散状蛋白条带,说明烘焙过程中蛋白质会发生聚集,同时也可能发生降解。对鲜花生Ara h 1条带的质谱分析结果显示,检测出70 条肽段,覆盖率达到79.2%;而烘焙后其中40 条肽段未能检出,但新增1 条肽段,且覆盖率降至43.9%。全部71 个肽段涉及到Ara h 1的18 个过敏原线性表位,酶解后鲜花生中检出16 个过敏原线性表位被破坏,烘焙花生中仅发现12 个被破坏。结论：烘焙加工会破坏蛋白质高级结构,掩盖了部分酶切位点,减少了酶解对过敏原线性表位的破坏,这可能是导致烘焙加工后Ara h 1致敏性强于未加工样品的原因。     

花生过敏原Ara h1免疫显性抗原决定簇的鉴别

为探索花生过敏原致敏机理,采用固相合成肽技术合成Ara h1的23条多肽。以花生过敏患者血清为抗体,鉴别和定位Ara h1的抗原决定簇。结果表明:Ara h1第21～34位,第89～98位,第393～403位,第498～507位,第594～605位氨基酸序列识别率在60%以上,为Arah1的抗原决定簇,其中第498～507位的多肽识别率为100%,是显性抗原决定簇,其氨基酸序列为RRYTARLKEG。用丙氨酸依次取代显性抗原决定簇的每个氨基酸,结果抗原决定簇的致敏性增强或丧失,说明Ara h1第499位和第503位的精氨酸和第502位的丙氨酸为降低Ara h1致敏性的关键氨基酸。     

核桃中主要过敏原Jug r 1的致敏性研究

近些年来,食物过敏的发病率呈逐年上升趋势。核桃是引起食物过敏的主要食品之一。目的:本文旨在研究核桃中主要过敏原Jug r 1与致敏性相关的免疫学特征,确定其线性抗原表位。方法:首先对核桃过敏原Jug r 1进行消化稳定性和热稳定性的研究,然后利用人体血清学分析其特异性抗体的结合情况,结合生物信息学对其氨基酸一级序列的相关特征参数的分析,预测其可能存在线性抗原表位,同时合成覆盖其全部氨基酸序列的重叠肽库,利用圆点免疫法(dot-blot)筛选出可与过敏患者血清中Ig E特异性结合的肽段,从而确定出Jug r1的线性抗原表位。结果 :核桃中主要过敏原Jug r 1在模拟胃液中60 min时仍未被完全消化;在90℃水浴中加热60 min其条带仍然存在,且可以与核桃过敏患者血清中的Ig E特异性结合。通过生物信息学软件和血清筛选出可结合的抗原表位。结论:核桃中主要过敏原Jug r 1具有较强的免疫学特性,且其存在3个免疫显性的抗原表位。对深入研究其过敏原改造和过敏机理提供理论依据。     

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)dihydrochloride,AAPH)和丙烯醛]对花生过敏蛋白Ara h 1的结构影响,进一步采用免疫印迹技术、酶联免疫法,模拟胃液消化和细胞模型分析其过敏原性的变化。结果 脂质过氧化物修饰后的花生过敏蛋白Ara h 1二级结构变得更无序,内源荧光强度降低;花生过敏蛋白Ara h 1的免疫球蛋白E结合能力、消化稳定性和释放活性介质能力均降低。结论 在花生加工过程中脂质过氧化物能够改变花生过敏蛋白的结构,降低其过敏原性。     

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。     

中国花生致敏蛋白的识别

我国对花生过敏方面的研究很少。本实验利用中国常用花生品种识别鉴定了中国主要的致敏蛋白,比较了国内外花生品种致敏蛋白相对含量的差异,期望找到中国花生过敏发病率较低的原因,为临床食物过敏患者的治疗和低过敏花生品种的培育提供理论依据。研究结果表明:Ara h1和三条Ara h3多肽是中国主要的致敏蛋白,并发现了Ara h1的亚基,分子量为58kD的多肽。Ara h1和Ara h3的相对含量各品种之间差异显著,并且低于国外花生品种。因此中国花生主要致敏蛋白相对含量低可能是导致中国花生过敏发病率较低的主要原因。     

花生过敏患者血清中抗Ara h1的IgG与IgE分离纯化研究

Ara h1是花生的一种主要致敏原蛋白,能够使花生过敏患者产生特异的IgG与IgE,从而导致花生过敏症状。Ara h1与其特异IgG和IgE的结合是导致花生致敏的一个重要原因。本研究利用Protein A亲和层析柱与Ara h1作为配体的亲和层析柱纯化抗Ara h1的IgG与IgE。高纯度的抗Ara h1特异IgG与IgE对探讨研究其与Ara h1之间相互作用具有重要意义。     

酪蛋白糖巨肽对花生过敏原免疫反应性的影响

目的 探究酪蛋白糖巨肽与花生过敏原相互作用并降低其免疫反应性的潜力。方法 通过蛋白-蛋白分子对接技术探讨酪蛋白糖巨肽(casein glycomacropeptides, CGMP)与Ara h1、Ara h2是否有相互作用的潜力。进一步通过混合水浴加热制备CGMP与花生蛋白的混合溶液(mixed solution of casein glycomacropeptides and peanut proteins, MCGP),建立MCGP致敏、花生蛋白激发的BALB/c小鼠模型,研究MCGP对花生过敏反应的影响。最后使用圆二色谱法研究酪蛋白糖巨肽与Ara h1、Ara h2的相互作用力及对其结构的影响。结果 CGMP与Ara h1、Ara h2间存在次级键(盐桥、氢键、范德华力),部分作用于过敏原表位;MCGP致敏组血清中的花生蛋白特异性免疫球蛋白E(Specific immunoglobulin E, sIgE)、sIgG₁、sIgG_2a含量显著下降,白介素-4(interleukin-4, IL-4)、IL-5、转化生长因子-β(transforming growth factor-β, TGF-β)、组胺水平显著下降,肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)水平显著升高, MCGP中Ara h2的α-螺旋与β-折叠的比例改变。结论 CGMP能够改变Ara h2的结构,遮蔽花生过敏原表位,抑制sIgE、sIgG结合Ara h1、Ara h2,降低部分花生过敏原的免疫反应性。     

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。     

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.     

Digestion of peanut allergens Ara h 1, Ara h 2, Ara h 3, and Ara h 6: A comparative in vitro study and partial characterization of digestion‐resistant peptides

Scope : There are differences in stability to pepsin between the major allergens in peanut; however, data are from different reports using different digestion models. This study provides a comprehensive comparison of the digestibility of the major peanut allergens. Methods and results : Peanut allergens Ara h 1, Ara h 2, Ara h 3 and Ara h 6 were incubated with pepsin to mimic the effect of gastric digestion. Samples were analyzed using SDS‐PAGE. To further investigate resistance to digestion, Ara h 2 was additionally subjected to digestion with trypsin and residual peptides were characterized. Ara h 1 and Ara h 3 were rapidly hydrolyzed by pepsin. On the contrary, Ara h 2 and Ara h 6 were resistant to pepsin digestion, even at very high concentrations of pepsin. In fact, limited proteolysis could only be demonstrated by SDS‐PAGE performed under reducing conditions, indicating an important role for the disulfide bridges in maintaining the quaternary structure of Ara h 2 and Ara h 6. Trypsin digestion of Ara h 2 similarly resulted in large residual peptides and these were identified. Conclusion : Ara h 2 and Ara h 6 are considerably more stable towards digestion than Ara h 1 and Ara h 3.     

花生过敏原Ara h7基因的克隆及序列分析

目的克隆花生Ara h7基因cDNA序列,并对其序列进行分析。方法采用Trizol法,从花生子叶中提取花生RNA,经反转录PCR,克隆花生Ara h7 cDNA序列;采用生物信息学软件对Ara h7序列进行分析,预测蛋白结构及功能。结果 Ara h7 cDNA序列有482 bp,编码160个氨基酸。生物信息学分析结果显示Ara h7蛋白是一种亲水性蛋白,分子量为18.881 kDa,具有9个潜在的磷酸化位点,5个不同的B细胞线性表位。结论本研究成功克隆了Ara h7 cDNA序列,并采用生物信息学软件分析了基因编码的蛋白的特征。     

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.     

Isolation and characterization of natural Ara h 6: evidence for a further peanut allergen with putative clinical relevance based on resistance to pepsin digestion and heat

Peanut allergy is a significant health problem because of its prevalence and the potential severity of the allergic reaction. The characterization of peanut allergens is crucial to the understanding of the mechanism of peanut allergy. Recently, we described cloning of the peanut allergen Ara h 6. The aim of this study was isolation and further characterization of nAra h 6. We purified nAra h 6 from crude peanut extract using gel filtration and anion exchange chromatography. The preparation was further characterized by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) with subsequent immunoblotting. Stability of nAra h 6 was studied by an in vitro digestibility assay as well as by resistance against thermal processing. Sequencing of nAra h 6 identified the N-terminal amino acid sequence as MRRERGRQGDSSS. Further results clearly demonstrated stability of nAra h 6 against pepsin digestion and heating. Immunoglobulin G (IgE) binding analysis and its biological activity shown by RBL 25/30-test of natural Ara h 6 supported the importance of this peanut allergen. Investigation of nAra h 6 revealed evidence for a further peanut allergen with putative clinical relevance based on resistance to pepsin digestion and heat.