【作者】 杨剑婷；

【导师】 吴彩娥；

【作者基本信息】 南京林业大学 ， 森林培育， 2010， 博士

【摘要】 论文以江苏泰兴产大佛指品种白果为原料,研究了白果蛋白的过敏性及致敏机理,为白果食品的安全评价及安全开发提供依据。主要研究结果如下:(1)通过原料处理方法及蛋白提取方法的比较,确定了白果蛋白粗提物的提取工艺。通过SDS-PAGE可知该白果蛋白物粗提物的主要蛋白有13条,其中以21 kDa和32 kDa的居多,pI为4.62,在检测方法极限内无氰化物检出。(2)以小鼠和豚鼠为受试动物,对白果蛋白粗提物进行了过敏性研究。经小鼠的急性毒性试验,在饲喂器能达到的蛋白最大浓度未见小鼠死亡。豚鼠给予白果蛋白粗提物后显过敏症状;对足垫激发有明显隆起;皮肤试验现阳性反应;血清的IgG和IgE水平均极显著高于对照(P<0.05)。组织病理学分析发现肺、肠和气管有炎症细胞浸润。综合分析,白果蛋白粗提物可致豚鼠发生过敏反应。(3)建立了白果蛋白过敏小鼠模型,并以此模型对致敏机理进行了研究。应用昆明种小白鼠,白果蛋白粗提液以100 mg/mL的浓度于第1、7、14 d经口灌胃致敏、以200 mg/mL的浓度于第21 d腹腔注射激发,致敏量和激发量都为0.3 mg/10g体重,可获得较好的过敏效果。白果蛋白处理后引起小鼠的IgE和IgG水平及血浆组胺含量显著升高(P<0.05),小鼠腹腔肥大细胞体外激发后脱颗粒,并释放出组胺,类胰蛋白酶活性也升高,肠、肝、肺部发生炎症症状,从而在体内诱发产生IgE抗体,并启动过敏介质的释放机制,导致过敏反应(Ⅰ型变态反应)的发生。(4)以白果蛋白粗提物为抗原,以经白果蛋白粗提物免疫的小鼠的血清为抗体,采用免疫印迹法进行特异性反应及与酶标二抗的反应,检测到白果蛋白粗提物中有3个蛋白(21 kDa、32 kDa、36 kDa)呈阳性反应。采用离子交换层析、凝胶层析,以SDS-PAGE检测,对其中一个蛋白进行了分离纯化研究,得到了32 kDa蛋白的纯化工艺和参数。该过敏蛋白分子量为32.12 kDa,是一个糖蛋白,蛋白与糖的比例为20.56:1。(5)从生理和生化的角度分析了白果过敏蛋白的氨基酸组成、紫外吸收光谱、糖肽键的类型,对热、酸、碱、盐和酶的稳定性。可知该蛋白耐热性较弱,耐酸碱性、耐盐性、耐消化性较强,是一个比较稳定的蛋白质,其稳定性可能也是导致其过敏的一个方面。(6)建立了间接ELISA法,以其测定经白果蛋白免疫后的过敏小鼠血清中IgE的效价为1:6400;建立了检测白果过敏原的双抗体夹心ELISA方法,白果蛋白的最低检出限为1.87μg/mL。更多还原

【Abstract】 Taking the ginkgo kernel, Dafozhi cultivar, harvested from Taixing, Jiangsu province, as material, allergicity and the mechanism of ginkgo protein were studied. The research would provide some basic materials for the ginkgo kernel safety assessment and product development. The main findings were as follows:(1) The extract technology of protein was conducted after the comparing of material treatment and extract methds. pI of the crude protein was 4.62. It was also ascertained no cyanide by assessing the possible toxins.(2) Allergicity of ginkgo kernel protein was studied in mice and guinea pigs. There was no death with the thickest concentration of protein in acute toxicity with mice. Guinea pigs were sensitized orally at days 1, 3 and 5, and challenged intraperitoneally. Allergy symptoms were showed after guinea pigs were treated with ginkgo kernel crude protein. Swelling in foot pads and a positive reaction emerged. IgE and IgG level from guinea pigs treated with ginkgo kernel protein were higher than those from CK, extremely significantly. There was inflammatory cell soakage. Taking into account, ginkgo kernel protein may be allergic to guinea pigs.(3) Ginkgo kernel protein allergy mouse model was evaluated and on the basis, allergy mechanism was studied. The established animal model was as follows. With Kunming mice, 100 mg/mLof ginkgo kernel protein extract was exposed orally at 1、7、14 d to sensitize and 200 mg/mL was treated intraperitoneallyto challenge with 0.3 mg/10g weight of both dose. According to this mouse model, animals was conducted as 4 groups, negative control (Tris-HCl buffer), ginkgo kernel protein of thin concentration, ginkgo kernel protein of thick concentration and positive control (OVA). The result showed IgE and IgG level of mice from each group during the immune time. The antibody level from 3 protein groups all was higher than the negative control significantly. After challenged in vitro, degranulation of mast cell was observed in 3 protein groups. Challenge in vitro also could result to histamine release typtase activity increased. Histamine content in plasma increased. Histopatholgic result indicated inflammatory manifestation in intestines, livers and lungs of mice from two ginkgo kernel protein groups. It could be deduced ginkgo kernel protein could resultⅠtype allergy reaction and the mouse model could be used in allergy researh. (4) Allergens of ginkgo kernel protein were detected with Western blotting. With ginkgo kernel proptein extract as antigen, sera from mice immunized with the protein extract as antibody, western blotting was producted. There were 3 proteins in the gel, and the molecular 21 kDa、32 kDa、36 kDa. The purification technology and parameters for 32 kDa were researched with ion exchange chromatography and gel chromatography, detected with SDS-PAGE. From it ginkgo kernel allergic protein from the above technology was a glycoprotein with molecular 32.12 kDa and the ratio of protein and sugar was 20.56:1.(5) Biochemistry and characteristics of the allergy protein were studied. The protein was not stable to heat, but stable in acid, alkali and salt solution, and anti-digested. Such stability perhaps resulted to its allergicity.(6) Undirective ELISA was used to detect the IgE titer of mice immunited with ginkgo kernel protein as 1:6400. Double-antibodies-sandwich-ELISA was established to assess antigen, with it, the least quantity of ginkgo kernel protein was 1.87μg/mL.更多还原