鸡蛋过敏原表位研究进展

鸡蛋过敏是一种常见的食物过敏反应,是人体对鸡蛋中蛋白质成分产生的一种变态反应。食物过敏反应的物质基础是抗原表位与抗体对位,解决食物过敏反应问题的关键一点即需要深入研究出过敏原的表位。本文介绍了鸡蛋中6种主要过敏原,简述了过敏原表位的定位方法,并详细综述了卵白蛋白、卵类粘蛋白、卵转铁蛋白和溶菌酶的过敏原表位研究进展。本文可为进一步鸡蛋过敏研究提供理论指导,也可为表位成分的定量检测以及无毒副作用鸡蛋过敏原疫苗的开发等方面提供一些参考。     

鸡蛋清中主要过敏原的研究进展

鸡蛋是引起食物过敏最常见的过敏原食物之一。本文重点介绍了鸡蛋清中主要过敏原卵类粘蛋白、卵白蛋白、卵转铁蛋白和溶菌酶的研究进展。     

色谱法在鸡蛋清主要过敏原分离纯化中研究进展

鸡蛋营养丰富,同时也是引起人体过敏的主要食物之一。蛋清中含有4种主要过敏原,包括卵白蛋白、卵转铁蛋白、卵类黏蛋白和溶菌酶。概述了鸡蛋中4种主要过敏原的理化特性及色谱法纯化过敏原的研究进展,旨在为规模化分离得到高纯度和高活性的过敏原提供参考。     

鸡蛋卵转铁蛋白过敏原B细胞线性表位的预测研究

为预测鸡蛋过敏原卵转铁蛋白的B细胞线性表位,以GenBank中提供的鸡蛋卵转铁蛋白氨基酸序列为基础,分别用Jameson-Wolf法、Kyte-Doolittle法、Emini法和Karplus-Schulz法对鸡蛋卵转铁蛋白的抗原指数、亲水性、蛋白表面可及性及柔韧性进行预测,并用Chou-Fasman法和Deleage-Roux法预测其二级结构,综合分析以上预测结果,得出鸡蛋卵转铁蛋白可能的B细胞线性表位区。结果表明鸡蛋卵转铁蛋白存在7个潜在表位区,包括蛋白第174～181、215～222、231～240、288～294、332～344、415～429、547～555位可能为B细胞线性表位优势区域。该结果将有助于鸡蛋卵转铁蛋白B细胞表位的进一步精确定位。     

一步法分离鸡蛋清中三种主要过敏原的研究

为快速高效分离鸡蛋中主要过敏原开展鸡蛋过敏的研究,本实验采用DEAE-Sepharose FF 色谱柱一步分离鸡蛋中的三种主要过敏原卵白蛋白、卵转铁蛋白和溶菌酶,通过SDS-PAGE 比对,纯度均高于相应标准品纯度,得率分别为87.66%、55.87% 和92.80%。本实验操作简便、设备简单、分离效率高,是一种分离鸡蛋过敏原的好方法。     

加工对鸡蛋过敏原的影响

卵类黏蛋白、卵白蛋白、卵转铁蛋白和溶菌酶是鸡蛋中的主要过敏原,适当的物理、化学和生物加工可以降低它们的致敏性。其中物理法生产的低致敏性蛋制品可供鸡蛋轻微过敏人群食用或作为免疫治疗药物;而化学和生物法制备的低过敏鸡蛋制品存在安全风险,它能否应用于生产,还有待进一步研究。另外,各种加工方法可以影响过敏原蛋白二硫键及高级结构甚至一级结构,从而导致该蛋白的致敏性发生变化。总之,加工对鸡蛋过敏原结构和致敏性的影响仍然是值得深入探索的科学问题,对指导生产和研发低致敏性或无致敏性蛋制品具有重要作用。     

卵转铁蛋白双抗夹心ELISA方法的建立

卵转铁蛋白是鸡蛋中的主要过敏原,针对鸡蛋中主要过敏原卵转铁蛋白建立了双抗夹心ELISA方法,该方法检出限为(15.37±1.20)ng/m L。除白蛋白外,该方法对粘蛋白、溶菌酶、α-乳白蛋白、牛奶总蛋白、β-乳球蛋白、酪蛋白、花生总蛋白、大豆、杏仁和绿豆等致敏蛋白几乎没有交叉反应。板内重复性和板间重复性较好,因此所建立的方法可用于对卵转铁蛋白的检测。     

腰果过敏原Ana o 2结构及抗原表位预测

腰果是引起人们过敏的主要食物之一。作者采用生物信息学方法,通过Pubmed网络服务器、生物信息分析软件SOPMA、swiss-model网络服务器、DNAStar生物分析软件等对腰果主要过敏原Ana o 2的结构和抗原表位进行预测,分析Ana o 2蛋白的抗原表位可能是108-111,181-186,217-218,234-238,244-255,283-287。这为腰果过敏原的进一步研究提供理论参考,并对开发低过敏腰果制品提供帮助。     

小鼠过敏模型在食品过敏原分析中的研究与应用

建立牛乳蛋白、河虾蛋白的ICR小鼠过敏模型,检测食品过敏原体外激发小鼠致肥大细胞的组胺释放率,探讨食品过敏原鉴定并验证所建过敏原体外评价新方法。方法以牛奶蛋白、河虾蛋白,氢氧化铝佐剂免疫ICR小鼠,建立IgE高反应性食物过敏动物模型。间接ELISA测定激发后第7、14、21d血清中过敏原sIgE水平;并在激发第14d分离小鼠腹腔肥大细胞,过敏原体外激发,紫外分光光度法检测类胰蛋白酶定向释放水平。①模型小鼠血清sIgE水平明显高于对照组,且在过敏激发后第14dsIgE效价最高,河虾组和牛奶蛋白组的sIgE效价分别达到1/25 600和1/12 800。②牛奶组类胰蛋白酶释放率达38.5%;河虾组的类胰蛋白酶释放率达47.1%,所有模型组与对照组间差异均达显著水平(P<0.05)。建立了一种小鼠过敏模型,通过检测过敏原体外激发类胰蛋白酶释放率可以作为一种鉴定与评价食品过敏原的有效方法。     

致敏小鼠血清代替过敏患者血清筛选Jug r 1 IgE线性抗原表位的可行性研究

目的:利用核桃过敏患者和核桃过敏原Jug r 1致敏小鼠血清筛选Jug r 1的IgE线性抗原表位,比较两者所筛选的表位的差异性,为今后利用小鼠血清替代人血清筛选食物过敏原表位提供理论依据,对食物过敏原研究及治疗具有重要意义。方法:利用固相合成肽法合成44个覆盖了Jug r 1一级序列的重叠短肽,通过圆点印迹试验利用核桃过敏患者血清和核桃过敏原Jug r 1致敏小鼠血清分别筛选出IgE抗原表位。结果:核桃过敏患者可以识别的IgE结合的线性抗原表位分别是肽段~1AALLVALLFVANAAA~(15)、4LVALLFVANAAAFRT18、~(16)FRTTITTMEIDEDID~(30)及~(125)CGISSQRCEIRRSWF~(139);核桃过敏原Jug r 1致敏小鼠所识别的抗原表位分别是1AALLVALLFVANAAA15和~(125)CGISSQRCEIRRSWF~(139)。结论:通过小鼠过敏血清筛选出的两个IgE线性抗原表位存在于过敏患者血清筛选出的4个IgE抗原表位中,提示在利用致敏小鼠血清代替过敏患者血清筛选IgE抗原表位时,可能出现假阴性结果,即有部分IgE线性抗原表位没有被识别。     

Preparation and Immunological Reactions of a Purified Egg Allergen Ovotransferrin

Ovotransferrin is one of the major allergens in egg white. To define the properties of ovotransferrin, a simple protocol for ovotransferrin purification was developed and a broad physicochemical and immunological assay were used for the characterization. The prepared ovotransferrin revealed a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and had an estimated purity of more than 97%. The identity of the prepared protein identified by mass spectrometry analysis demonstrated that it was iron-free ovotransferrin with a molecular weight of 77,726 Da and pI value of 6.85, respectively, and its nature folded structure was kept well as defined by circular dichroism spectroscopy. Western blotting and enzyme-linked immunosorbent assays showed strong immune reactions between the prepared ovotransferrin and rabbit polyclonal antibody against ovotransferrin/egg-allergic patients' sera, suggesting that the prepared ovotransferrin bound to IgG and IgE strongly. In addition, 24 of the potential linear IgE binding epitopes on the prepared ovotransferrin were predicted using a web-based allergen database for food safety. The preparation method of ovotransferrin in the present work might provide scientific support to the authenticity of ovotransferrin for developing certified reference materials.     

The panel of egg allergens, Gal d 1-Gal d 5: Their improved purification and characterization

Egg proteins represent one of the most important sources evoking food allergic reactions. In order to improve allergy diagnosis, purified and well-characterized proteins are needed. Although the egg white allergens Gal d 1, 2, 3 and 4 (ovomucoid, ovalbumin, ovotransferrin, and lysozyme) are commercially available, these preparations contain impurities, which affect exact in vitro diagnosis. The aim of the present study was to set up further purification protocols and to extend the characterization of the physicochemical and immunological properties of the final batches. The egg white allergens Gal d 1-4 were purified from commercial preparations, whereas Gal d 5 (alpha-livetin) was purified from egg yolk. The final batches of Gal d 1-5 consisted of a range of isoforms with defined tertiary structure. In addition, the IgE binding capacity of the purified egg allergens was tested using allergic patients' sera. The allergen batches will be further used to set up allergen specific diagnostic assays and to screen a larger collection of patients' sera.     

Immunoreactivity of hen egg allergens: Influence on in vitro gastrointestinal digestion of the presence of other egg white proteins and of egg yolk

Hen egg white comprises of a complex mixture of proteins, which greatly differ in their physicochemical characteristics and relative abundance. We aimed to identify potential undiscovered egg allergens within the egg white proteome and investigated the existence of matrix effects on the proteolytic stability and resultant IgE-binding of the allergenic proteins. In addition to the main egg allergens: ovalbumin (OVA), ovomucoid (OM) and lysozyme (LYS), two minor egg white proteins, tentatively identified as ovoinhibitor and clusterin, were found to react with serum IgE from egg-allergic patients. Egg white exhibited residual immunoreactivity after gastrointestinal digestion due to the presence of intact OVA and LYS, as well as of several IgE-binding peptides derived from OVA. The presence of egg yolk slightly increased the susceptibility to hydrolysis of egg white proteins and abrogated bile salt-induced precipitation of LYS in the duodenal medium. However, the resultant immunoreactivity against IgE of egg white proteins after in vitro digestion was not significantly modified by the presence of yolk components.     

热加工对鸡蛋中4种主要过敏原结构的影响

为探究热加工对鸡蛋主要过敏原结构的影响,对鸡蛋清中的4种过敏原进行不同条件的热加工,并利用圆二色光谱、紫外-可见光光谱、外源荧光光谱对加热前后过敏原的空间结构进行表征。结果表明,卵转铁蛋白对热不稳定,随加热时间的延长和温度的升高,分子结构逐渐展开,芳香族残基和疏水基团暴露于分子表面;轻微加热使卵白蛋白分子的二级结构更加有序,吸光度减小,表面疏水性增大,随着加热程度的增大,其二级结构变得无序,分子发生聚集;加热后卵类黏蛋白的二级结构较为稳定,轻微加热其分子展开,吸光度和荧光强度增大,继续加热使蛋白分子部分聚集;热处理使溶菌酶分子的有序性下降,芳香族残基和疏水基团逐渐暴露,但随着加热程度的增加,蛋白分子发生部分聚集。本实验结果为后续研究加热对鸡蛋过敏原致敏性的影响提供理论依据。     

Comparative resistance of food proteins to adult and infant in vitro digestion models

IgE‐mediated allergy to milk and egg is widespread in industrialised countries and mainly affects infants and young children. It may be connected to an incomplete digestion of dietary proteins causing an inappropriate immune response in the gut. In order to study this, a biochemical model of infant gastroduodenal digestion has been developed, which has reduced levels of protease (eightfold for pepsin and tenfold for trypsin and chymotrypsin), phosphatidylcholine and bile salts, compared with the adult model. This model has been used to study the behaviour of three characterised food‐relevant proteins (bovine β‐lactoglobulin (β‐Lg), β‐casein (β‐CN) and hen's egg ovalbumin), all of which are relevant cows' milk and hens' egg allergens. Digestion products were characterised using electrophoresis, immunochemical techniques and MS. These showed that ovalbumin and β‐CN were digested more slowly using the infant model compared with the adult conditions. Resistant fragments of β‐CN were found in the infant model, which correspond to previously identified IgE epitopes. Surprisingly, β‐Lg was more extensively degraded in the infant model compared with the adult one. This difference was attributed to the tenfold reduction in phosphatidylcholine concentration in the infant model limiting the protective effect of this phospholipid on β‐Lg digestion.     

Changes in the antigenic and immunoglobulin E-binding properties of hen's egg albumin with the combination of heat and gamma irradiation treatment

This study was carried out to evaluate the changes in the allergenic and antigenic properties of hen's egg albumin (ovalbumin [OVA]) with the combination of heat and gamma irradiation treatment. OVA solution samples were treated by (i) heating (sample 1), (ii) irradiation after heating (sample 2), and (iii) heating after irradiation (sample 3). Samples were isothermally heated and irradiated at the absorption dose of 10 kGy. Competitive indirect enzyme-linked immunosorbent assays (ELISAs) were performed with blood serum to test the ability of treated OVA to bind to immunoglobulin E (IgE) and mouse murine monoclonal antibody (IgG). OVA's ability to bind to mouse IgG changed upon heating at 75 degrees C, and its ability to bind to egg-allergic IgE changed upon heating at 80 degrees C. The ELISAs showed that egg-allergic IgE did not recognize OVA very well when heated at > or = 80 degrees C, while mouse IgG retained better activity under these conditions. Egg-allergic IgE binding was low both for OVA samples treated by heating and for samples treated by irradiation followed by heating. These results show that allergies induced by OVA could be effectively reduced by the combination of heat and gamma irradiation treatment.