腰果过敏原Ana o 3蛋白的分离纯化及热稳定性研究

Ana o 3蛋白是腰果主要的过敏原之一。以生腰果为原料,通过粉碎脱脂、低盐提取粗蛋白、冷冻干燥、阴离子交换层析及超滤浓缩等过程分离得到目的蛋白,利用小分子蛋白电泳、液相色谱-串联质谱和免疫印迹技术进行鉴定;此外,采用紫外光谱及圆二色光谱分析腰果过敏原Ana o 3蛋白在160℃持续加热10、15、20、25、30min的结构变化,以评估分离纯化后腰果过敏原Ana o 3蛋白的稳定性。结果表明,成功建立了一种快速高效纯化腰果过敏原Ana o 3蛋白的方法,该方法获得的蛋白纯度高于95%。通过圆二色光谱发现腰果过敏原Ana o 3蛋白在160℃加热过程中α-螺旋构象逐步转变为β-折叠构象,β-转角含量变化较小,无规则卷曲含量稍有增加,表明该蛋白二级结构较为稳定;经紫外光谱发现加热后腰果过敏原Ana o 3蛋白的紫外特征吸收峰的吸光度升高,表明加热会使腰果过敏原Ana o 3蛋白变性,结构展开,更多的色氨酸和酪氨酸残基暴露,使得空间结构变得松散;经蛋白电泳和免疫印迹分析发现,热处理后腰果过敏原Ana o 3蛋白会发生降解,表位被破坏。希望研究可为高纯度腰果过敏原Ana o 3蛋白的纯化提供方案,为腰果过敏的研究提供基础,为腰果过敏原在热加工过程中的稳定性研究提供理论依据。     

食品中杏仁过敏原成分的检测

目的：建立检测食品中桃仁、杏仁过敏原成分的荧光PCR 方法,比较国外3 种ELISA 试剂盒效果。方法：针对杏仁Pru du1 基因设计引物及探针,建立荧光PCR 方法。利用杏仁过敏原参考物质对3 个品牌的ELISA试剂盒的回收率进行比较。结果：建立的荧光PCR 方法,具有很好的特异性;灵敏度为10mg/kg。结论：桃仁及杏仁过敏原成分荧光PCR 检测方法特异性好、灵敏度高,对食品中过敏原的检测有重要的实际意义。     

花生过敏原蛋白分离纯化方法研究进展

花生中已确定的过敏原蛋白包括Ara h 1～Ara h 11 11种。本文详细介绍花生中主要过敏原蛋白(Ara h 1、Ara h 2、Ara h 3/4、Ara h 6)以及非主要过敏原蛋白(Ara h 7～Ara h 11)的分离纯化方法研究进展。花生过敏原蛋白的分离纯化方法包括硫酸铵沉淀法、柱层析法、电泳法。其中硫酸铵沉淀法主要用于粗提纯化过程,而柱层析法则主要用于花生过敏原蛋白的精制,它包括离子交换层析、凝胶过滤层析、亲和层析、疏水相互作用层析、高效液相色谱。目前离子交换层析和凝胶过滤层析在花生过敏原蛋白分离纯化中应用最为广泛,而电泳法则仅见应用于Ara h 7及油质蛋白(Ara h 10、Ara h 11)的分离纯化。     

鲤鱼小清蛋白过敏原的分离纯化

以普通鲤鱼为材料,采用磷酸盐粗提、及离子交换层析的方法,纯化出鲤鱼中主要过敏原——鲤鱼小清蛋白。结果显示,采用阴离子交换层析方法制取的鲤鱼小清蛋白纯度达90%以上,该方法为过敏原鲤鱼小清蛋白的分离研究提供了可行的实验参数。     

三文鱼过敏原小清蛋白分离纯化 及免疫结构鉴定

小清蛋白是鱼类的主要过敏原,三文鱼小清蛋白具有不同的亚型,分子量相近,序列基本相同,给分离纯化带来了不小的挑战。本试验采用硫酸铵盐析、Qxl-Sepharose离子交换结合凝胶过滤等方法定向纯化小清蛋白β1型,并应用免疫印迹法对纯化的小清蛋白进行过敏原性鉴定,结合激光辅助解析/飞行时间质谱进行结构鉴定。结果显示,硫酸铵盐析、离子交换层析结合凝胶过滤纯化方法可以得到纯度达90%以上的小清蛋白β1型,且蛋白具有很强的免疫反应活性。针对该蛋白的研究不仅有利于过敏原检测方法的建立,也可为低致敏性水产品的开发提供理论依据。     

凡纳对虾原肌球蛋白硫酸铵沉淀分离纯化方法的优化

虾原肌球蛋白(tropomyosin,TM)食物过敏是一种全球性的食品安全问题,研究发现TM的分离纯化对于系统准确地鉴定和控制过敏具有重要的意义,鉴于此,本研究以南美白对虾(Litopenaeus vannamei)为对象,改进了TM分离纯化的方法。结果表明,抽提液p H 6.5~7.5、硫酸铵饱和度30%时,可以有效分离纯化TM;经喹啉酸法测定其质量浓度最高可达43μg/μL。该方法省去了高效液相色谱、层析技术等对TM的进一步纯化,减少了纯化步骤,避免了在这些纯化步骤中出现的蛋白损失及蛋白稀释;高质量浓度的TM溶液可无须经过冷冻干燥,直接用于对其理化性质及过敏原性的研究。     

牛奶过敏原的分离、鉴定与纯化

对牛奶过敏原进行分离、鉴定与纯化。通过SDS-PAGE电泳分离牛奶的蛋白质组份,采用免疫印迹(Western-blotting)方法鉴定过敏原,通过离子交换层析对牛奶过敏原进行初步纯化。结果表明,鲜牛奶粗提液SDS-PAGE显示蛋白条带有12条,奶粉粗提液SDS-PAGE显示出的蛋白条带与鲜牛奶的蛋白条带基本一致。鲜牛奶Western-Blotting显示14ku的阳性条带。离子交换层析可初步纯化出14ku的过敏原蛋白。本研究对牛奶过敏原进行了分离和鉴定,并初步纯化出牛奶的主要过敏原。     

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 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。     

鲤鱼小清蛋白的纯化及其过敏原性鉴定

目的：分离纯化鲤鱼小清蛋白(parvalbumin),并对其进行过敏原性鉴定,为建立鱼类过敏原检测技术奠定基础。方法：采用硫酸铵分级盐析和阴离子交换层析纯化鲤鱼小清蛋白,采用点杂交和特异性IgE 检测试剂盒筛选鱼类过敏者血清,应用聚丙烯酰胺凝胶电泳和免疫印迹技术分析确定纯化目标蛋白的性质。结果：硫酸铵分级盐析和阴离子交换层析纯化方法可以得到电泳纯单一目标蛋白;小鼠抗蛙小清蛋白单克隆抗体免疫印迹实验表明,所得纯化蛋白是小清蛋白。此外,免疫杂交结果显示,鱼类过敏者血清能与纯化的小清蛋白发生特异性结合,从而证实了鲤鱼小清蛋白的过敏原性。     

Quantification of Peach Fruit Allergen Lipid Transfer Protein by a Double Monoclonal Antibody-based Sandwich ELISA

Fruit is one of the most commonly reported food allergy sources in China, and peach lipid transfer protein (Pru p 3) has been identified as the major allergen inducing systematic symptoms. Crude allergen extracts and single component allergens have been used in food allergy diagnosis and immunotherapy. Reliable and sensitive analytical methods to quantify Pru p 3 content in fruit will help to identify low-allergenic cultivars among the abundant peach genetic resources in China. In this study, we developed a sensitive sandwich ELISA method to measure Pru p 3 in peach fruit based on two monoclonal antibodies with high sensitivity and linearity. Significant variability was observed in peach cultivars, with a much higher Pru p 3 level in peach peel than in pulp. This method will be very useful to select peach varieties with low Pru p 3 content and as a diagnostic product to detect hidden allergens in processed food.     

Purification and structural stability of the peach allergens Pru p 1 and Pru p 3

Pru p 1 (a Bet v 1 homologue) and Pru p 3 (a nonspecific lipid transfer protein; nsLTP) are major allergenic proteins in peach fruit, but differ in their abundance and stability. Pru p 1 has low abundance and is highly labile and was purified after expression as a recombinant protein in Escherichia coli. Pru p 3 is highly abundant in peach peel and was purified by conventional methods. The identities of the proteins were confirmed by sequence analysis and their masses determined by MS analysis. The purified proteins reacted with antisera against related allergens from other species: Pru p 1 with antiserum to Bet v 1 and Pru p 3 with antiserum to Mal d 3 (from apple). The presence of secondary and tertiary structure was demonstrated by circular dichroism (CD) and high field NMR spectroscopy. CD spectroscopy also showed that the two proteins differed in their stability at pH 3 and in their ability to refold after heating to 95 degrees C. Thus, Pru p 1 was unfolded at pH 3 even at 25 degrees C but was able to refold after heating to 95 degrees C at pH 7.5. In contrast, Pru p 3 was unable to refold after heating under neutral conditions but readily refolded after heating at pH 3.     

Extraction and mass spectrometry identification of a major peach allergen Pru p 1

BACKGROUND: Peach allergy can be caused by the allergen Pru p 1. This occurs by cross‐reactivity with the homologous birch pollen allergen Bet v 1. However, the direct identification of Pru p 1 as an immunoglobulin E (IgE)‐binding protein extracted from peach fruit has never been reported. RESULTS: Phosphate‐buffered saline (PBS) and phenol extractions were applied to solubilise the proteins from peach peel and pulp, and IgE immunoblotting with sera of individual peach‐allergic patients was used to detect the potential allergens. Most of the patients showed binding to an 18 kDa band in IgE immunoblotting performed with the phenolic extracts of peach peel and pulp, but not when the PBS extracts were used. Mass spectrometry of the 18 kDa spot excised from a two‐dimensional electrophoretic gel showed this protein to correspond to the peach allergen Pru p 1. CONCLUSION: Phenol extraction was necessary to detect by IgE immunoblotting a major peach allergen, which showed very low extractability with PBS, indicating the appropriateness of adopting different extraction procedures to identify plant allergens. The 18 kDa peach protein was definitively identified as the Bet v 1‐homologous peach allergen Pru p 1. Copyright © 2011 Society of Chemical Industry     

Examining the effect of High Pressure Processing on the allergenic potential of the major allergen in peach (Pru p 3)

The aim of this work is to study the effect of High Pressure Processing (HPP) on the allergenicity of the main protein involved in peach allergy (Pru p 3). Results obtained showed that most pressure/time combinations slightly enhanced in vitro IgE-binding to Pru p 3 and peach extract. Moreover, additional tests were carried out by means of skin prick tests on peach allergic patients. Different from in vitro results, in vivo effects evidenced that HPP (600 MPa/5 min) can either reduce or increase Pru p 3 allergenicity, depending on the particular sensitisation of each patient. Notwithstanding this variability, it is highly remarkable that the skin response to pressurized peach extract was stronger in more than half of individuals. These results would suggest a higher risk of HPP-treated peach products to elicit an allergenic reaction. However, it has been also proved that matrix plays an important role in peach allergenicity modification. Consequently, further investigations are needed before extrapolating results to more complex products.Industrial relevancePeach allergy is one of the most frequent allergies in Mediterranean countries among adult patients. However, up to date, there is no information about the effect of HPP on peach allergenicity. This study increases understanding about the impact of processing on an important fruit allergen in order to advance knowledge-based ways to managing allergens risks in industry, as well as it opens new opportunities of research in other technologies or strategies in the attempt to reduce peach allergenicity.     

Identification of four IgE-reactive proteins in raspberry (Rubus ideaeus L.)

IgE-reactive proteins in raspberry (Rubus ideaus L.) were identified using PCR, RT-PCR, 2-DE and MS/MS peptide sequencing. Specific polyclonal antibodies and patient sera were used in Western blotting to identify crossreactive epitopes. Initially, two potential allergens Rub i 1 and Rub i 3 were detected using PCR, showing high sequence identity to proteins in Rosaceous species like Mal d 1 and Mal d 3 from apple, Pru av 1 and Pru av 3 from cherry and Pru p 1 and Pru p 3 from peach. Furthermore, de novo identified peptides of a protein band at about 30 kDa reacting with most of the patient sera tested (> 80%) revealed a high sequence homology with class III chitinases. Raspberry chitinase, when subjected to glycoproteomic analysis, showed typical complex plant-type N-glycans with a core alpha1,3 fucose and a beta1,2 xylose at least at one position, indicating the presence of crossreacting carbohydrate determinants (CCDs). Finally, MS/MS analysis revealed an IgE-reactive raspberry cyclophilin, homologous to Bet v 7. Results obtained suggest that the consumption of raspberries might be responsible for adverse reactions in sensitised individuals.     

Peel LTP (Pru p 3) – the major allergen of peach – is methylated. A proteomic study

Lipid transfer protein (LTP, Pru p 3) is the major allergen of peach (Prunus persica), and is in a greater abundance in the peel than in the pulp of the fruit. Peel LTP is more allergenic than pulp LTP, but it is not clear whether this is due to its specific allergenic properties or to its higher concentration.     

Almond allergens: update and perspective on identification and characterization

Almond (Prunus dulcis) is not only widely used as a human food as a result of its flavor, nutrients, and health benefits, but it is also one of the most likely tree nuts to trigger allergies. Almond allergens, however, have not been studied as extensively as those of peanuts and other selected tree nuts. This review provides an update of the molecular properties of almond allergens to clarify some confusion about the identities of almond allergens and our perspective on characterizing putative almond allergens. At present, the following almond allergens have been designated by the World Health Organization/International Union of Immunological Societies Allergen Nomenclature Sub-Committee: Pru du 3 (a non-specific lipid transfer protein 1, nsLTP1), Pru du 4 (a profilin), Pru du 5 (60S acidic ribosomal protein 2), Pru du 6 (an 11S legumin known as prunin) and Pru du 8 (an antimicrobial protein with cC3C repeats). Besides, almond vicilin and almond γ-conglutin have been identified as food allergens, although further characterization of these allergens is still of interest. In addition, almond 2S albumin was reported as a food allergen as a result of the misidentification of Pru du 8. Two more almond proteins have been called allergens based on their sequence homology with known food allergens and their ‘membership’ in relevant protein families that contain allergens in many species. These include the pathogenesis related-10 protein (referred to as Pru du 1) and the thaumatin-like protein (referred to as Pru du 2). Almonds thus have five known food allergens and five more likely ones that need to be investigated further. Published 2020. This article is a U.S. Government work and is in the public domain in the USA.     

Improving In Vitro Detection of Sensitization to Lipid Transfer Proteins: A New Molecular Multiplex IgE Assay

Scope

LTP-syndrome is characterized by sensitization (IgE) to multiple non-specific lipid transfer proteins (nsLTPs) with a variable clinical outcome. The treatment is primarily based on offending food avoidance. However, the determination of Pru p 3-specific IgE is currently the main diagnostic tool to assess sensitization to nsLTPs. Herein, the study evaluates improvement of LTP-syndrome diagnosis and clinical management using a new IgE multiplex-immunoblot assay with a high diversity of food nsLTPs.

Methods and results

An EUROLINE-LTP strip with 28 recombinant nsLTPs from 18 allergenic sources is designed. In total the study investigates 38 patients with LTP-syndrome and compares results from the nsLTPs (LTP-strip) with the respective food extracts of Prick-by-prick (PbP) testing. The agreement exceeds 70% for most nsLTPs, e.g., Pru p 3 (100%), Mal d 3 (97%), Pru av 3 (89%), Pha v 3 isoforms (87%/84%), Ara h 9 (82%), Cor a 8 (82%), and Jug r 3 (82%). The functionality and allergenic relevance of nine recombinant nsLTPs are proven by Basophil activation testing (BAT).

Conclusions

The new IgE multiplex-immunoblot nsLTP assay shows a good diagnostic performance allowing culprit food assessment. Negative results from LTP-strip may indicate potentially tolerable foods, improving diet intervention and patients’ quality of life.     

桃花中营养及功能性成分分析

目的对不同颜色桃花的营养物质和功能性成分进行分析测定,以明确桃花的化学组成。方法对桃花水提液、乙醇提取液和石油醚提取液中组分进行系统定性鉴定,并在此基础上对白色、粉色、红色三种桃花中的蛋白质、还原糖、总酚、总黄酮、花色苷含量和氨基酸及酚类物质组成进行分析。结果桃花中含有糖、蛋白质(氨基酸)、有机酸、酚类物质、游离黄酮及其苷类、皂苷、生物碱等,其中总糖含量为31.99%~34.53%,还原糖含量为18.20%~24.44%,可溶性蛋白含量为6.48%~8.19%,氨基酸含量为9.68%~12.02%,总酚含量为7.30%~9.03%,总黄酮含量为3.89%~4.69%,粉色和红色桃花中含有花色苷,含量为2.35%~9.29%,白色桃花不含花色苷;不同种类桃花具有不同的氨基酸组成和酚类物质组成。结论桃花富含营养物质与功能性成分,是一种很有开发利用价值的植物资源。