低敏花生品种筛选及电子束辐照对其免疫原性影响的研究

发布时间：2019-03-17 20:26

【摘要】：食物过敏反应(foodhypersensitivity,FH)也称为食物变态反应(foodallergy)，是一种过度的免疫反应，严重的可致死亡。随着当代食品加工和国际贸易的发展，国内外过敏性疾病的发病率在逐年增加，对居民的正常生活造成了严重的影响。作为我国主要油料作物的花生是八大主要过敏食物之一，其免疫原性严重威胁着人体的健康。治疗食物过敏疾病的唯一有效方法就是严格控制食物过敏原，寻求降低或根除食物免疫原性的方法，已成为食品安全研究的关键之一。本论文从电子加速器降低花生的免疫原性，和从我国丰富的花生种质资源中筛选低免疫原性或无过敏性的花生品种两个方面进行了研究。本文首先确定了花生过敏原粉的制备方法，即经过正己烷脱脂后，采用磷酸盐直接浸提法提取蛋白质，超声波破碎细胞壁提高蛋白提取率，将离心得到的过敏原液透析，冷冻干燥得过敏原粉，冷冻保存。结合我国花生过敏患者血清，初步鉴定了我国的主要过敏原为Arah1,Arah2和Arah3。收集了33种中国花生和23种日本花生，进行花生品种间免疫原性差异性比较的研究。利用聚丙烯酰胺凝胶电泳和凝胶成像系统分析了花生的主要过敏原的种类及其含量，采用间接ELISA试验探讨了各品种间免疫原性的高低。发现，绥中大花生和沭阳大站秧缺失Arah3（35，，35kDa），新金伏大、兴城大花生、如东碗儿青、日本品种JP-16和JP-19缺失Arah3（24kDa）。56个品种中Arah1的相对含量都是最高的，中国的在25~47.6%范围内，日本的在20.2~39.6%范围内，其次是Arah2（20kDa）和Arah3（14kDa）的相对含量。通过间接ELISA试验，结合中国花生过敏患者的血清，得出中国花生的免疫原性较日本花生的强。中国花生品种中免疫原性最低的是20号铁岭四粒红，最高的是19号营口四粒红。日本的花生JP-10的免疫原性最强，JP-6的免疫原性最低。花生的免疫原性取决于非常复杂的因素，单纯的过敏条带的缺失或含量的高低和花生的其免疫原性没有直接关系。本试验研究了电子束辐照降低花生过敏原免疫原性的效果及对其生化性质的影响。选择2、4、 6、8、10、15、20kGy剂量的电子束辐照对花生过敏原液和脱脂花生粉。辐照后花生主要过敏原（Arah1,Arah2,Arah3）蛋白分子质量的变化变化用SDS-聚丙烯酰胺凝胶电泳测定，免疫原性的变化用免疫印迹和间接竞争ELISA法测定。辐照后花生过敏原溶液的浓度、浊度及疏水性的变化用紫外分光光度计和荧光光度计分别进行测定。结果表明，花生过敏原在溶液状态比固体状态对辐照处理更为敏感。当辐照剂量低于10kGy时处理的过敏原液的免疫原性有稍微的增强，剂量大于10kGy时免疫原性降低，剂量为20kGy时过敏原液的IC50值是未处理的11倍。辐照使过敏液的浓度和浊度随着辐照剂量的加大而增加，疏水性与对照组相比增强，当辐照剂量大于15kGy时又降低。电子束辐照改变了过敏原的生化性质，降低了其免疫原性，比较而言，辐照对液体状态下过敏原的影响更显著。综上，本文建立了花生过敏原粉制备的方法，初步鉴定了主要过敏原，筛选出缺失过敏原条带的品种，证实了电子束辐照降低免疫原性可行性。
[Abstract]:Food allergy (FH), also known as food allergy, is an excessive immune response that can cause death. With the development of modern food processing and international trade, the incidence of allergic diseases at home and abroad has increased year by year, which has a serious effect on the normal life of the residents. As the main oil crop in China, the peanut is one of the eight major allergic foods, and its immunogenicity is a serious threat to the health of the human body. The only effective way to treat food allergy is to strictly control the food allergen, seek to reduce or eliminate the immunogenicity of food, and has become one of the key to food safety research. In this paper, the immunogenicity of peanut was reduced from the electron accelerator, and the two aspects of low immunogenicity or non-allergic peanut varieties were selected from the abundant peanut germplasm resources in China. The preparation method of peanut allergen powder is firstly determined, namely, after defatting with n-hexane, the protein is extracted by adopting a phosphate direct extraction method, the cell wall of the ultrasonic wave is broken to improve the protein extraction rate, the allergen liquid obtained by centrifugation is dialyzed, and the allergen powder is obtained by freeze-drying to obtain the allergen powder and the freezing protection The main allergens in our country were Ara1, Ara2 and Aah in combination with the serum of peanut allergy patients in China. 3.33 Chinese and 23 Japanese peanuts were collected, and the difference of immunogenicity among the peanut varieties was compared. The types and contents of the main allergens of the peanut were analyzed by means of the gel-gel electrophoresis and gel-imaging, and the immunogenicity of the main allergens was studied by indirect ELISA. The high and low levels of Arah3 (35,35 kDa), New Gold-Volta, large peanut in Xingcheng, such as Eastern and Erqing, Japanese variety JP-16 and JP-19, Arah3 (24 kDa) were found. The relative contents of Ara1 in 56 varieties were the highest, and in China in the range of 25-47.6%, Japan was 20.2-39.6%. Phase, followed by the phase of Ara2 (20 kDa) and Arah3 (14 kDa) To determine the immunogenicity of peanut in China by indirect ELISA and in combination with the serum of Chinese peanut allergy patients. The lowest immunogenicity of Chinese peanut varieties is that of the No.20 Tieling Red, the highest of which is Yingkou, No.19. Four red. Japanese peanut JP-10 has the strongest immunogenicity, and the immunogen of JP-6 The lowest sex is that the immunogenicity of the peanut is dependent on the very complex factors, the absence or content of the simple allergic band and the immunogenicity of the peanut are not straight The effect of electron beam irradiation on the immunogenicity of peanut allergen and its biochemical properties were studied in this paper. The effect of. Select 2,4,6,8,10,15,20 kGy dose of electron beam irradiation to peanut allergen liquid and Degreasing peanut powder. The change of the molecular weight of the main allergen (Ara1, Ara2, Arah3) in the peanut after irradiation was determined by SDS-polyacrylamide gel electrophoresis. The change of immunogenicity was determined by immunoblotting and indirect competition ELI. Determination of the concentration, turbidity and hydrophobicity of peanut allergen solution after irradiation with ultraviolet spectrophotometer and fluorometer The results show that the peanut allergen is irradiated at the irradiation position in the solution state than the solid state It is more sensitive. When the irradiation dose is less than 10 kGy, the immunogenicity of the treated allergen liquid is slightly enhanced, the immunogenicity is reduced when the dose is greater than 10 kGy, and the IC50 value of the allergen liquid at a dose of 20 kGy is not The concentration and turbidity of the allergic liquid increased with the increase of the irradiation dose, and the hydrophobicity was enhanced as compared with the control group. When the irradiation dose was more than 15kG The electron beam irradiation changes the biochemical properties of the allergen, reduces the immunogenicity, compared with the allergen in the liquid state by irradiation, In this paper, the method of the preparation of peanut allergen powder was established, and the main allergens were identified and screened out. The decrease of the electron beam irradiation was confirmed by the variety of the allergen strip.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R155.5

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