Development of an innovative immunoassay for CP4EPSPS and Cry1AB genetically modified protein detection and quantification

An innovative immunoassay, called enzyme-linked immunoabsorbant assay (ELISA) Reverse, based on a new conformation of the solid phase, was developed. The solid support was expressly designed to be immersed directly in liquid samples to detect the presence of protein targets. Its application is proposed in those cases where a large number of samples have to be screened simultaneously or when the simultaneous detection of different proteins is required. As a first application, a quantitative immunoassay for Cry1AB protein in genetically modified maize was optimized. The method was tested using genetically modified organism concentrations from 0.1 to 2.0%. The limit of detection and limit of quantitation of the method were determined as 0.0056 and 0.0168 (expressed as the percentage of genetically modified organisms content), respectively. A qualitative multiplex assay to assess the presence of two genetically modified proteins simultaneously was also established for the case of the Cry1AB and the CP4EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) present in genetically modified maize and soy, respectively.     

转基因食品潜在致敏性评价方法的研究进展

随着转基因食品在人类消费生活中的应用日益增加,人们对于完善转基因食品潜在致敏性评价方法的需求也不断增强.本文就现有转基因食品潜在致敏性的评价方法,包括决策树、序列同源性、血清学筛选、消化稳定性(抗胃蛋白酶性)以及动物模型等的研究进展做一概述,对其现状做出总结并对其发展提出建议.     

不同酶水解对鸡蛋清蛋白降解和潜在致敏性的影响

酶水解是降低食物过敏原致敏性的一种常用手段,本文分别利用胃蛋白酶、木瓜蛋白酶、中性蛋白酶和碱性蛋白酶水解鸡蛋清蛋白,通过三羟甲基氨基甘氨酸-十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（Tricine-SDS-PAGE）,并结合水解度（邻苯二甲醛法）分析监测蛋清蛋白的酶解过程,进一步利用制备的兔抗蛋清蛋白多克隆抗体血清和鸡蛋过敏患者血清池评估酶解产物的抗原性和致敏性。结果表明:木瓜蛋白酶和碱性蛋白酶能够有效的水解蛋清蛋白,并且所得酶解产物的抗原性和致敏性较低,其中,木瓜蛋白酶水解蛋清蛋白后产物的抗原性降低了59.23%,致敏性降低了4.91%;碱性蛋白酶水解蛋清蛋白后产物的抗原性降低了57.61%,致敏性降低了4.55%。因此,木瓜蛋白酶和碱性蛋白酶对鸡蛋清蛋白降解及致敏性降低方面均有显著影响。      

Effects of gastrointestinal digestion and heating on the allergenicity of the kiwi allergens Act d 1, actinidin, and Act d 2, a thaumatin-like protein

Kiwifruit is a significant elicitor of allergy both in children and adults. Digestibility of two kiwifruit allergens, actinidin (Act d 1) and thaumatin-like protein (Act d 2), was assessed using an in vitro digestion system that approximates physiological conditions with respect to the passage of food through the stomach into the duodenum. Act d 1 precipitated in simulated gastric fluid at pH 2 and digestion of the aggregated protein proceeded slowly. The residual precipitate redissolved completely in simulated duodenal fluid at pH 6.5 and was partially digested. Forty percent of Act d 2 remained intact during gastric digestion and were cleaved by duodenal proteases into large fragments covalently linked by disulfide bonds. Both digested allergen samples displayed nearly unchanged IgE binding abilities. Circular dichroism spectra were used to analyze heat and acid-induced unfolding. Thermal stability of both allergens was strongly pH dependent. While Act d 1 was irreversibly destabilized in acidic solutions, heat-induced denaturation of Act d 2 at pH 2 was fully reversible. IgE binding to Act d 2 but not Act d 1 was detected in processed food products. The stability of Act d 1 and Act d 2 provides one explanation for the allergenic potency of kiwifruit.     

利用牛奶过敏者血清检验牛奶中过敏原酶解程度的研究

利用对牛奶过敏者和对牛奶不过敏者的血清作为“一抗”,采用酶联免疫吸附实验(ELISA)的方法,检测牛奶水解后对牛奶过敏者血清的结合能力,从而确定牛奶中过敏原蛋白的最佳水解消除条件,降低牛奶及奶制品对牛奶过敏者的致敏性,达到牛奶过敏人群可以饮用的标准。结果表明:胃蛋白酶、胰蛋白酶和木瓜蛋白酶共同水解牛奶的最佳条件是酶与底物质量比W(E/S)=4%,pH1.5(胃蛋白酶)和pH8.0(胰蛋白酶和木瓜蛋白酶),温度40℃(胃蛋白酶和胰蛋白酶)和45℃(木瓜蛋白酶),水解时间3h,用酶标仪在492nm测得其水解后奶样与过敏者血清结合的OD值已降到了牛奶不过敏者血清与未水解牛奶结合的OD值水平,达到了消除过敏原的效果。     

转双反义淀粉分支酶基因大米中蛋白质的营养价值评价

目的 对转双反义淀粉分支酶基因大米和亲本大米蛋白质的营养价值进行评价.方法 采用通用的非生物学评价方法,即化学评分、氨基酸评分、蛋白质消化率校正后的氨基酸评分、必需氨基酸指数、营养指数、氨基酸比值系数和氨基酸比值系数分,评价大米蛋白质的营养价值.结果 转基因大米和亲本大米的化学评分分别为62,59;氨基酸评分分别为75,62;蛋白质消化率校正后的氨基酸评分分别为65,56;必需氨基酸指数分别为79,80;营养指数分别为11,8;氨基酸比值系数分分别为76.7,67.0;氨基酸比值系数都是l.1.结论 转双反义淀粉分支氨基因大来中抗性淀粉含童显著提高,但并未影响到其自身蛋白质的营养价值.该转基因大米蛋白质的营养价值与亲本大米具有"实质等同性".     

Effect of inulin on the rheological properties of silken tofu coagulated with glucono-δ-lactone

This study investigated the rheological properties of inulin-containing silken tofu coagulated with glucono-δ-lactone (GDL) upon heating. Inulin (Raftiline^® HP-gel) was added to a soy protein isolate-enriched cooked soymilk at 0%, 1%, 2%, 3% and 4% (w/v) levels along with 0.4% (w/v) GDL to prepare acid-induced silken tofu. Gelation was induced by heating the soymilk mixture from 20 to 90 °C at a constant rate (1 °C/min) or isothermally at 90 °C for 30 min. The gelling properties were measured with dynamic small-deformation mechanical analysis and static large-deformation compression tests. The rheological changes in soymilk during gelation were dependent upon both the pH decline (hydrolysis of GDL) and the specific temperature of heating. Control samples heated to 50 °C, with the pH lowered to 5.95, started to gel, showing a rapid increase in storage (G′) and loss (G″) moduli afterwards. The addition of 2% inulin lowered the on-set gelling temperature by 2.8 °C and improved (P < 0.05) both rheological parameters of the tofu gel as well as hardness and rupture force (textural profile analysis) of the formed silken tofu. The results indicated that inulin enhances the viscoelastic properties of GDL-coagulated silken tofu, and the textural effect of inulin is an added benefit to its current application mainly as a prebiotical ingredient in food.