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%。
Little information was so far available about allergenic mechanism of the roasted peanut allergens during initial stages of allergy.The purpose of this study was to determine the influence of roasting(150℃,20 min)on biochemical and biological properties of Ara h 3,a major peanut allergen.Allergenicity of roasted peanut emulsion to mice,differences in uptakes between Ara h 3 purified from raw peanuts(named as Ara h 3-Raw)and that purified from roasted peanuts(named as Ara h 3-Roasted)by bone marrow-derived dendritic cells(BMDCs)and the implication of cell surface receptors involving in uptake,and changes in glycosylation and structure of Ara h 3 after roasting were analyzed in this study.This study suggested that roasting increased allergenicity of peanut to BALB/c mice.Maillard reaction and structural changes of Ara h 3 induced by roasting significantly altered the uptake of Ara h 3-Roasted by BMDCs,and modified Ara h 3 fate in processes involved in immunogenicity and hyper allergenicity,indicating that food processing pattern can change food allergenicity.
基于花生主要过敏原Ara h 1的基因序列设计引物,采用复合引物技术构建竞争性扩增内标,以157copies/PCR为内标添加量,建立含扩增内标的花生过敏原PCR检测方法。结果表明,该方法的检测灵敏度为0.1ng DNA,特异性良好;将该方法应用于9种食品的花生过敏原成分检测,结果与标签标识一致,表明该方法具有一定的应用价值。
从花生中提取总RNA,用反转录聚合酶链式反应得到花生过敏原Ara h 1基因,构建pET-28a-Ara h 1表达载体,转入Rosetta(DE3)宿主表达菌中诱导产物表达,用镍离子亲和层析法纯化得到目的蛋白。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示目的蛋白分子质量约为75 kDa,与预计相符;经质谱鉴定为Ara h 1蛋白。用BALB/c小鼠模型评价重组Ara h 1蛋白的致敏性结果显示,重组Ara h 1蛋白致敏小鼠血清中特异性抗体、Th2型细胞因子、组胺含量升高,空肠和肺组织发生病变,表明重组Ara h 1蛋白可以导致小鼠发生Th2型过敏反应,且有与天然Ara h 1蛋白相似的致敏性。同时RBL细胞模型结果显示重组Ara h 1蛋白还可导致RBL细胞脱颗粒,释放β-己糖苷酶,进一步表明重组Ara h 1蛋白具有致敏性。
