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。     

花生中主要过敏原的研究进展

花生是影响部分人群生活质量的过敏原食物之一。本文重点介绍了花生中主要过敏原Arah1、Arah2、Arah3的研究进展。     

花生中主要过敏原的研究进展

花生是影响部分人群生活质量的过敏原食物之一。本文重点介绍了花生中主要过敏原Arah1、Arah2、Arah3的研究进展。      

花生Ara h6基因的克隆、表达以及免疫活性鉴定

目的:克隆花生主要过敏原Arah6基因,诱导表达并纯化该蛋白,检测其免疫活性。方法:提取花生总RNA,设计特异性引物,RT-PCR克隆花生Arah6的基因,将测序正确的片段连入原核表达载体pET-28a上,并转入BL21（DE3）宿主表达菌中,IPTG诱导表达,通过Ni2+亲和层析柱纯化目的蛋白,Western-blotting检测该重组蛋白的免疫原性。结果:测序结果表明花生Arah6目的片段全长为438bp,编码145个氨基酸,与GenBank中蛋白序列100%相同。该基因诱导表达的产物纯化后经SDS-PAGE鉴定为17kDa。Western-blotting结果表明该蛋白与花生过敏病人混合血清中IgE结合,具有免疫原性。结论:成功克隆花生过敏原Arah6的基因,该基因表达的重组蛋白具有良好的免疫原性。      

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。      

花生内源过敏原蛋白Arah2的分离纯化及鉴定

Arah2蛋白是花生的主要过敏原蛋白之一,为获得高纯度的花生Arah2蛋白,以新鲜花生为原料,通过蛋白浸提、DEAE-Sepharose Fast Flow阴离子交换层析,十二烷基磺酸钠—聚丙烯酰胺凝胶电泳(SDS—PAGE)回收等方法分离得到目的蛋白,并运用基质辅助激光解吸/电离飞行时间质谱(MALDI—TOF/MS)对其进行鉴定。结果表明:该方法可得到纯度较高的纯化蛋白;经质谱鉴定后确定该蛋白为Arah2蛋白,两个同种异型物分子量分别为18.5 kD(Arah2.01)和20.1kD(Arah2.02);层析柱分离和SDS—PAGE电泳回收的得率分别为31.4%和18.6%。     

基于Arah6的花生问接竞争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（折合成舍花生的含量,约为165ng／mL～100μg／mL）,抑制方程为抑制率I=21．4181gC-6．0633（c为Amh6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。     

多重PCR同时检测常见8种食物过敏原

依据大豆Lectin基因,小麦Gliadin基因,花生Arah3基因,腰果Ana o3基因,鱼和虾16S rRNA,牛和鸡线粒体DNA设计特异性引物序列.在单一PCR方法基础上,建立2种4重PCR方法检测8种食物过敏原的技术.该方法检测周期短,具有较好的特异性和灵敏性,可用于对食品中多种食物过敏原的检测和监控.     

花生过敏原Ara h2.02原核表达方法条件的研究

将重组质粒pET-32a(+)-Arah2.02转化表达宿主菌Rosetta(DE3)中,经IPTG诱导表达,SDS—PAGE电泳分析,结果显示表达的蛋白大小约为38kDa。进一步用通用His标签抗体进行Western Blotting检测,结果表明成功克隆表达了花生过敏原Arah2.02。为获得较多的重组蛋白Arah2.02,分别对IPTG浓度、摇床转速、诱导温度和时间等条件进行选择,确定最佳条件为:IPTG浓度0.3mmol/L,摇床转速220r/min,诱导温度37℃,诱导时间2h。     

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Arah1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。     

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。     

IDENTIFICATION AND QUANTIFICATION OF MAJOR PEANUT ALLERGENS (IN CHINA) WITH IGE-BINDING PROPERTY

Peanut allergens have not been studied in China. This study aimed to investigate (1) whether there are differences in the relative amounts of major peanut allergens between the Chinese peanut varieties and the American, and (2) the effect of cooking methods on peanut allergenicity. The allergenic property of raw peanuts and peanut preparations was assessed by immunoblotting and enzyme-linked immunosorbent assay. The relative contents of the major peanut allergens were quantified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis densitometry. For results, Ara h 1 and Ara h 3 were major peanut allergens in China. The amounts of Ara h 1 and Ara h 3 in peanut varieties differ significantly and were both lower than the American varieties. The immunoglobulin E (IgE)-binding ability of different processed peanuts to IgE was not significantly different. Therefore, peanut varieties may induce different amounts of allergens. The relative lower contents of Ara h 1 and Ara h 3 may lead to the lower prevalence of peanut allergy.

PRACTICAL APPLICATIONS

Because of its nutritional and rheological properties, peanut is used in a wide range of different foods, and peanut allergy represents an important health problem. It is essential to identify the compounds of peanut allergens and to study their characteristics in order to explore approaches for the therapies and to breed the nonallergenic peanut seed.     

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。     

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。     

Enzymatic treatment of peanut kernels to reduce allergen levels

This study investigated the use of enzymatic treatment to reduce peanut allergens in peanut kernels as affected by processing conditions. Two major peanut allergens, Ara h 1 and Ara h 2, were used as indicators of process effectiveness. Enzymatic treatment effectively reduced Ara h 1 and Ara h 2 in roasted peanut kernels by up to 100% under optimal conditions. For instance, treatment of roasted peanut kernels with α-chymotrypsin and trypsin for 1–3 h significantly increased the solubility of peanut protein while reducing Ara h 1 and Ara h 2 in peanut kernel extracts by 100% and 98%, respectively, based on ELISA readings. Ara h 1 and Ara h 2 levels in peanut protein extracts were inversely correlated with protein solubility in roasted peanut. Blanching of kernels enhanced the effectiveness of enzyme treatment in roasted peanuts but not in raw peanuts. The optimal concentration of enzyme was determined by response surface to be in the range of 0.1–0.2%. No consistent results were obtained for raw peanut kernels since Ara h 1 and Ara h 2 increased in peanut protein extracts under some treatment conditions and decreased in others.     

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.     

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.     

Polyphenol oxidase/caffeic acid may reduce the allergenic properties of peanut allergens

Polyphenol oxidase (PPO) catalyzes the oxidation of tyrosine residues of proteins and, therefore, their cross‐linking. Previously we demonstrated that cross‐links produced by peroxidase (POD), which also catalyzes tyrosine oxidation, led to a reduction in the allergenic properties of peanut allergens. 11 We postulated in this study that PPO can also reduce the allergenic properties by cross‐linking the allergens. Because caffeic acid, a phenolic compound, can cross‐link proteins, its effect on peanut allergens was also examined. In the experiments, peanut extracts were treated with and without PPO, PPO/caffeic (pH 8, 37 °C for 1 h) and caffeic acid (pH 10.5, overnight), respectively. The samples were then analyzed for cross‐links and IgE binding by SDS‐PAGE, Western blots, and competitive inhibition ELISA. Results showed that, in all cases, cross‐links and a decrease of the levels of two peanut major allergens, Ara h 1 and Ara h 2, were observed. Of the three treatments, PPO/caffeic was the most effective in reducing IgE binding or the allergenic properties of peanut allergens. The availability of tyrosine residues was also demonstrated in a POD‐treated system, using a monoclonal antibody against dityrosine. We concluded that PPO/caffeic acid reduced the allergenic properties of Ara h 1 and Ara h 2 by cross‐linking and decreasing the levels of allergens. Copyright © 2005 Society of Chemical Industry     

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.