辐照对Pen a1抗原表位免疫原性的影响

辐照处理刀额新对虾中主要过敏原Pen a1,采用SDS-PAGE,MOLDI-TOF MS方法分析辐照处理对虾致敏蛋白Pen a1分子质量的影响,同时利用表位抗体进行免疫印迹及间接竞争ELISA法评估Pen a1中5种抗原表位免疫原性的变化情况。结果显示:与未处理相比,6.7 k Gy辐照处理能够破坏部分Pen a1蛋白结构,导致裂解和聚合现象的发生。5种抗原表位免疫原性均未降低,并有不同程度的增加,其中Epitope1,2,3,4的IC50值约为对照的4/5,Epitope5的IC50值变化不大,说明其对辐照处理较稳定。低剂量的辐照处理不能降低Pen a1抗原表位的免疫原性。     

榛子主要过敏原Cor h 1单克隆抗体识别抗原表位区域的确定

目的 确定已获得的四株榛子主要过敏原Cor h 1单克隆抗体识别的抗原表位区域。方法 构建谷胱甘肽疏基转移酶 (GST)与Cor h 1不同截短体的表达载体并表达GST-Cor h 1融合蛋白, 采用ELISA和Western blot检测单抗与不同融合蛋白的反应, 并结合DNAstar表位分析软件推知不同单抗的抗原识别表位区域。结果 确定了四株Cor h 1单抗的抗原识别表位区域, 分别是4G7和2H3抗体的抗原表位位于Cor h 1第120~126位氨基酸, 5F2和1G4抗体的抗原表位位于Cor h 1第43~52位氨基酸。结论 四株Cor h 1单抗的抗原识别表位区域的确定, 为Cor h 1的进一步研究和食品安全检测奠定了基础。     

虾过敏原Troponin C (Pen m6)的克隆表达、免疫学鉴定及生物信息学分析

目的对斑节对虾(Penaeus monodon)第6组过敏原(Pen m6),肌钙蛋白C(Troponin C)进行克隆表达、免疫学鉴定并研究其生物学意义。方法提取斑节对虾总RNA;根据Gen Bank:HM034316.1设计引物及构建表达载体PET-24a-Pen m6;转化至大肠杆菌BL21(DE3)进行表达;纯化后的重组Pen m6用Western bolt来鉴定免疫学特性;用生物信息学相关工具对Pen m6进行同源性分析,并预测其蛋白质的结构和功能。结果克隆出斑节对虾Pen m6基因开放阅读框453 bp,编码150个氨基酸。重组Pen m6蛋白呈可溶性,分子量约35kDa,能够与斑节对虾过敏患者血清IgE结合。斑节对虾与凡纳滨对虾亲缘关系比较近,其中斑节对虾Pen m6蛋白与凡纳滨对虾Troponin C1(gb|AET36896.1|)同源性为98%。理化性质预测Pen m6蛋白质不稳定。蛋白质结构预测结果显示Pen m6的结构主要以α螺旋组成。T细胞抗原表位预测得到4个肽序列(15~23、35~43、91~99、112~120)。B细胞抗原表位预测得到4个肽序列(7~16、21~30、28~37、58~67)。结论克隆的重组斑节对虾Pen m6蛋白具有良好的免疫原性,为进一步研究斑节对虾过敏原的结构成分及其理化性质奠定理论基础。     

芝麻过敏原Ses i 3同源建模及B细胞线性抗原表位预测

为预测芝麻过敏原Ses i 3的B细胞线性抗原表位和三级结构,使用SOPMA、DNAStar和The BepiPred1.0 Server等生物信息学软件,采用多种方案对Ses i 3抗原性指数、亲水性、表面可及性、柔韧性等参数及其二级结构进行预测,对预测抗原表位综合分析。利用SWISS-MODEL程序进行同源建模并用Ramachandran软件评价结构稳定性。结果:序列SESKDP,RQKHQGEHG,NRKSP,QHG,YQREKGRQDDDNPTDPEKQY,RRQG,KYREQQGREGGRGE,EGR,EQGR,QHG,RQDR,ENP,RHE,ESK,RPTH,ASQ,SRSRGSYQGETRGRP,ANNNE,SRSQQ,GPRQQQQGR最可能是Ses i 3蛋白的B细胞线性抗原表位。以5e1r.2为模板,同源建模的方式成功构建了芝麻过敏原Ses i 3蛋白的三级结构,并经Ramachandran评价显示蛋白模型构象稳定。此结果为制备特异性抗体肽段及过敏原检测等提供了依据。     

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。     

饼干模型对小麦及花生过敏原消化稳定性和免疫活性的影响

食品加工或食物基质可以不同程度地影响过敏原消化稳定性和免疫原性。然而,对食品加工和食物基质对 食物模型中过敏原的影响却知之甚少。本实验通过体外模拟胃肠消化的方式,包括模拟口腔咀嚼、胃部消化和十二 指肠消化,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和免疫印迹的方法,分析焙烤模型饼干中小麦过敏原和花生 过敏原的消化特性和免疫原性。结果显示：小麦和花生蛋白均可被胃蛋白酶迅速水解,醇溶蛋白、谷蛋白等致敏原 被降解成低分子质量多肽;可溶性蛋白中花生过敏原Ara h 1和Ara h 3基本消失,Ara h 2/6耐受胃肠消化;酶联免疫 吸附测定结果显示,消化后饼干中过敏原的致敏性降低。综合以上结果表明,饼干模型的消化性质基本不受焙烤加 工和其他基质的影响,免疫原性因致敏原被消化而降低。     

苯甲酸人工抗原的合成与免疫效价的测定

通过苯甲酸的衍生物邻苯二甲酸酐(PA)的氨解法将苯甲酸(BA)偶联到载体蛋白上合成苯甲酸的人工抗原:免疫原苯甲酸-鸡卵清蛋白(BA-OVA)和包被原苯甲酸-牛血清白蛋白(BA-BSA).通过紫外光谱扫描、红外光谱扫描及SDS聚丙烯酰胺凝胶电泳的方法检测鉴定人工抗原,并用免疫原(BA-OVA)免疫BALB/c小鼠,检测抗体的生成.结果表明,苯甲酸人工抗原的合成成功.根据邻苯二甲酸酐、人工抗原和载体蛋白的紫外吸收值,计算出免疫原中苯甲酸(BA)与载体鸡卵清蛋白(OVA)的偶联比约为174:1.免疫小鼠血清中的抗体通过ELISA法检测为阳性(血清效价达到了1:3.2×10<'5>),表明合成的抗原已诱导出针对苯甲酸的特异性抗体生成,为进一步的苯甲酸单克隆抗体制各和免疫检测提供可靠的免疫原.     

食用油中苯并(a)芘的快速检测研究

采用间接免疫法结合显色反应快速检测食用油中苯并(a)芘,在抗体与磁珠上苯并(a)芘抗原结合的基础上,通过辣根过氧化酶连接苯并(a)芘二抗进行间接免疫反应。结果表明:优化的免疫磁珠反应条件为苯并(a)芘抗体用量4μg,辣根过氧化酶用量100μg,免疫反应温育时间60min;方法的线性范围为0. 5 pg/m L～500 ng/m L,检出限为0. 12μg/kg,定量限为0. 5μg/kg,加标回收率为89. 3%～100. 1%。方法具有良好的特异性和稳定性,符合食品污染物现场快速检测的基本要求。     

大豆球蛋白A3酸性多肽的原核表达及B细胞表位分析

人工合成大豆球蛋白A3酸性多肽基因cDNA序列,构建原核表达载体pET30a-A3,将重组质粒转化大肠杆菌(E.coli)BL21后用IPTG诱导表达,经His亲和层析纯化,获得纯度约91%融合A3蛋白。Western blotting显示,所获得的A3融合蛋白可与对豆粕过敏的仔猪血清发生免疫反应,表明所得融合蛋白具有免疫活性。利用Lasergene7.0中Protean软件对A3蛋白氨基酸序列进行B细胞抗原表位预测和分析,推测其中97～113、156～160、169～176、186～193、271～284和295～312氨基酸区段是A3酸性多肽的B细胞抗原表位,其中169～176区段的平均抗原指数最高,表位抗原性最强。本结果为进一步研究该蛋白致敏机理及单抗制备奠定了初步基础。     

鸡蛋过敏人群中主要过敏原的反应性及卵类粘蛋白抗原表位的初步研究

目的分析年龄、性别对鸡蛋过敏人群中主要过敏原反应性的影响,初步探索我国鸡蛋过敏儿童卵类粘蛋白的主要抗原表位。方法以卵类粘蛋白、卵白蛋白、卵转铁蛋白纯品为抗原,采用免疫斑点印迹,检测21例鸡蛋过敏儿童血清特异性免疫球蛋白E(SIgE)与蛋白反应性,分析年龄、性别因素对过敏原反应性的影响;应用DNAStar生物分析软件,预测并合成卵类粘蛋白抗原表位肽段,采用免疫斑点印迹检测抗原表位与鸡蛋过敏儿童血清的反应性。结果 A(≤1岁)组、B组(2~3岁包括3岁)、C组(3岁)的卵类粘蛋白反应率差异有统计学意义(P0.05),卵白蛋白、卵转铁蛋白反应率各组间均差异无统计学意义(P0.05);性别对各过敏原的反应率差异无统计学意义(P0.05);卵类粘蛋白抗原表位反应率较低。结论卵类粘蛋白sIgE在不同年龄段存在差别,卵白蛋白sIgE在21例患者均可检测出,卵转铁蛋白s Ig E在各年龄段无明显差别;应用DNAStar生物软件预测的卵类粘蛋白抗原表位,在天津地区反应性低,不是该地区主要抗原表位。     

基于质谱检测转基因生物外源蛋白质的消化稳定性

基于无标记定量质谱检测技术建立了一种新的转基因生物外源蛋白质消化稳定性评价方法。以牛β-乳球蛋白、牛血清白蛋白和大豆胰蛋白酶抑制剂为标准蛋白质,经模拟人体胃/肠消化液消化后进行凝胶电泳分离,切取目标蛋白质条带进行酶切,提取肽段进行纳升液相色谱-电喷雾串联质谱分析。基于Mascot数据库检索鉴定目标蛋白质。计算各消化时间点蛋白质匹配肽段数与消化前蛋白质匹配肽段数的比值,当比值≤0.50时判断为目标蛋白质在该时间段内已消化。将此方法应用于转基因抗虫水稻“华恢1号”外源蛋白Cry1Ab/1Ac的消化稳定性分析,结果显示在模拟胃液中消化2 min时,比值下降到0.50以下,在模拟肠液中消化15 s后比值下降到0.50以下,表明该蛋白在胃/肠消化液中具有消化不稳定性。     

Identification of immunoglobulin E epitopes on major allergens from dairy products after digestion and transportation in vitro

Dairy processing can alter the digestion stability and bioavailability of cow milk proteins in the gastrointestinal tract. However, analysis of stable linear epitopes on cow milk allergens that could enter into intestinal mucosal is limited. Thus, this study aimed to investigate the digestion and transportation properties and residual allergen epitopes entering into gastrointestinal mucosa of 3 commercial dairy products, including pasteurized milk (PM), ultra-heat-treated milk (UHTM), and dried skim milk (DSM). In this work, the digestive stability of the 3 kinds of dairy products has been performed in a standard multistep static digestion model in vitro and characterized by Tricine-SDS-polyacrylamide gel electrophoresis and reversed-phase HPLC. With respect to gastrointestinal digestion in vitro, the main allergens including β-lactoglobulin (β-LG), α-lactalbumin (α-LA), and caseins were degraded gradually, and the resistance peptides remained in the PM with a molecular weight of range from 3.4 to 5.0 kDa. Simultaneously, the potential allergenicity of the cow milk proteins was diminished gradually and is basically consistent after 60 min of gastrointestinal digestion. After gastrointestinal digestion, the remaining peptides were transported via an Ussing chamber and identified by liquid chromatography-MS/MS. By alignment, 10 epitopes peptides were identified from 16 stable peptides, including 5 peptides (AA 92–100, 125–135, 125–138, and 149–162) in β-LG, 2 peptides in α-LA (AA 80–93 and 63–79), 2 peptides in α_S1-casein (AA 84–90 and 125–132), and 1 peptide (AA 25–32) in α_S2-casein were identified by dot-blotting mainly exist in UHTM and PM. This study demonstrates dairy processing can affect the digestion and transport characteristics of milk proteins and in turn alter epitope peptides release.     

Bioinformatic screening and detection of allergen cross‐reactive IgE‐binding epitopes

Protein allergens can be related by cross‐reactivity. Allergens that share relevant sequence can cross‐react, those lacking sufficient similarity in their IgE antibody‐binding epitopes do not cross‐react. Cross‐reactivity is based on shared epitopes that is based on shared sequence and higher level structure (charge and shape). Epitopes are important in predicting cross‐reactivity potential and may provide the potential to establish criteria that identify homology among allergens. Selected allergen's IgE‐binding epitope sequences were used to determine how the FASTA algorithm could be used to identify a threshold of significance. A statistical measure (expectation value, E‐value) was used to identify a threshold specific to identifying cross‐reactivity potential. Peanut Ara h 1 and Ara h 2, shrimp tropomyosin Pen a 1, and birch tree pollen allergen, Bet v 1 were sources of known epitopes. Each epitope or set of epitopes was inserted into random amino acid sequence to create hypothetical proteins used as queries to an allergen database. Alignments with allergens were noted for the ability to match the epitope's source allergen as well as any cross‐reactive or other homologous allergens. A FASTA expectation value range (1 × 10^?5–1 × 10^?6) was identified that could act as a threshold to help identify cross‐reactivity potential.     

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。     

Resistance of casein-derived bioactive peptides to simulated gastrointestinal digestion

The resistance of six casein-derived peptides, including antihypertensive peptides RYLGY, AYFYPEL and YQKFPQY, to simulated gastrointestinal digestion and the effect on angiotensin-converting enzyme (ACE)-inhibitory activity were evaluated. After digestion, peptides RYLGY, AYFYPEL, and YQKFPQY were partly hydrolysed by the digestive enzymes. RYLGY and AYFYPEL maintained potent ACE-inhibitory activity, with IC₅₀ values as low as 9.3 and 4.7 μg mL⁻¹, respectively. Digestion fragments were sequenced and then synthesised to evaluate their activity. Several showed potent ACE-inhibitory activity, which could explain the in vitro activity of the digests. A notable antioxidant activity was also observed. Since AYFYPEL was less susceptible to digestion, we focused on the antihypertensive activity in spontaneously hypertensive rats of the main digestion fragments of RYLGY. Interestingly, these peptides showed moderate effects in vivo. This suggests that the undigested fraction could also contribute to the in vivo effects of RYLGY and AYFYPEL, and other minor fragments may also participate.     

Genetic variants of bovine β- and κ-casein result in different immunoglobulin E-binding epitopes after in vitro gastrointestinal digestion

Immunoglobulin E-mediated allergy to cow milk is a common allergy in industrialized countries, mainly affecting young children and infants. β-Casein (CN) and κ-CN belong to the major allergens in cow milk. Within these milk proteins, genetic polymorphisms occur, which are characterized by substitutions or deletions of AA, resulting in different variants for each protein. Until now, these variants have not been considered when discussing the allergenic potential of bovine milk. In this study, the focus was placed on the arising peptide pattern after in vitro gastrointestinal digestion of several β- and κ-CN variants to determine resistant fragments containing IgE-binding epitopes and to identify potential differences between these variants. β-Casein A¹, A², and B, as well as κ-CN A, B, and E, were separated and isolated from milk of cows homozygous for these variants and digested with an in vitro gastrointestinal digestion model. The resulting peptides were identified using mass spectrometry and compared with previously determined epitopes. Seven β-CN and 4 κ-CN peptides, common in all β- or κ-CN variants, remained of sufficient size to harbor IgE-binding epitopes. In addition, some peptides and, consequently, epitopes differ from each other due to the AA substitution occurring in the individual variants. The distinct peptides AA 108 to 129 of β-CN A¹ and A², AA 103 to 123 of β-CN B, as well as AA 59 to 72, AA 59 to 80, and AA 58 to 80 of all 3 β-CN variants correspond to the IgE-binding epitopes AA 107 to 120 and AA 55 to 70, respectively. In κ-CN, the 2 variant-specific peptides AA 136 to 149 (κ-CN A, E) and AA 134 to 150 (κ-CN B) are congruent with the IgE-binding epitope AA 137 to 148. The present study shows that genetic polymorphisms affected the arising peptide pattern of the caseins and thus modifications in the IgE-binding epitopes occurred. As a consequence, the casein variants could show differences in their allergenicity. Studies investigating the allergenic potential of these different peptides are currently in progress.     

In vitro digestion and characterisation of 2S albumin and digestion‐resistant peptides in pecan

The 2S albumins are one of the major protein families involved in severe food allergic reactions to nuts, seeds and legumes, thus potentially making these proteins clinically relevant for allergic sensitisation and potential diagnostic markers. In this study, we sought to purify native 2S albumin protein from pecan to further characterise this putative allergen. The purified 2S albumin, Car i 1, from pecan was found to be resistant to digestion by pepsin in simulated gastric fluid (SGF) and comparatively stable to proteolysis by trypsin and pancreatin in simulated intestinal fluid (SIF). Digestion of purified Car i 1 in SGF and SIF resulted in formation of different digestion‐resistant peptides that were capable of binding IgE antibodies from allergic individuals. Digestion stability of Car i 1 and formation of digestion‐resistant antigenic peptides may explain why it is a potent sensitising protein in pecan for susceptible individuals. The observation that digestion‐resistant peptides are able to bind IgE implies that pecan can trigger systemic allergic reactions even after digestion in the stomach and small intestine.     

转高赖氨酸融合蛋白基因水稻蛋白消化稳定性

以我国自主培育的转高赖氨酸融合蛋白基因水稻(简称转GL基因水稻)为材料,参考中华人民共和国国家标准,通过体外模拟胃/肠液消化实验,研究转GL基因水稻蛋白的消化稳定性。结果表明：质量浓度为5g/L的总蛋白在胃液中2min内完全消化,质量浓度为2g/L的总蛋白在肠液中15s～2min内完全消化;而以5倍于国标规定的质量浓度进行消化时,总蛋白在模拟胃液中60min仍可观察到未降解片段,在模拟肠液中2min内则全部消化;两步法体外模拟消化结果,胃液中极难降解的蛋白片段在经肠液作用5min后,完全被降解。本研究表明,转GL基因水稻蛋白在模拟胃肠液中不具有消化稳定性。     

Comparative study of in vitro digestibility of major allergen,tropomyosin and other proteins between Grass prawn (Penaeus monodon) and Pacific white shrimp (Litopenaeus vannamei)

BACKGROUND: Stability in simulated gastric fluid is supposed to be an important parameter for the estimation of food allergenicity. In the present study, the digestive stability of allergenic protein tropomyosin (TM) and other food proteins from Grass prawn and Pacific white shrimp in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) digestion assay system was investigated and comparatively studied by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‐PAGE), western blotting, and inhibition enzyme‐linked immunosorbent assay (ELISA). RESULTS: In the SGF system, proteins such as actin and myosin heavy chain (MHC) were rapidly degraded within a short period of time, while TM was relatively resistant to pepsin digestion. In the SIF system, MHC was also easily decomposed, while TM and actin were resistant to digestion. Western blotting using a specific polyclonal antibody against TM indicated that the degradation pattern of shrimp TM by SGF and SIF was almost unaffected by the presence of other myofibrillar proteins. Further study by IgE immunoblotting and inhibition ELISA using sera from crustacean‐allergic patients indicated that IgE binding of TM was decreased. CONCLUSION: Proteinase digestion is effective in reducing IgE binding of shrimp TM. It is also of interest to notice that Pacific white shrimp TM had higher digestion stability than Grass prawn TM. However, Pacific white shrimp TM revealed enhanced IgE binding over that of TM from Grass prawn and thus it is possibly more allergenic. Copyright © 2010 Society of Chemical Industry