低敏花生品種篩選及電子束輻照對(duì)其免疫原性影響的研究
發(fā)布時(shí)間：2019-03-17 20:26
【摘要】：食物過敏反應(yīng)(foodhypersensitivity,FH)也稱為食物變態(tài)反應(yīng)(foodallergy)，是一種過度的免疫反應(yīng)，嚴(yán)重的可致死亡。隨著當(dāng)代食品加工和國(guó)際貿(mào)易的發(fā)展，國(guó)內(nèi)外過敏性疾病的發(fā)病率在逐年增加，對(duì)居民的正常生活造成了嚴(yán)重的影響。作為我國(guó)主要油料作物的花生是八大主要過敏食物之一，其免疫原性嚴(yán)重威脅著人體的健康。治療食物過敏疾病的唯一有效方法就是嚴(yán)格控制食物過敏原，尋求降低或根除食物免疫原性的方法，已成為食品安全研究的關(guān)鍵之一。本論文從電子加速器降低花生的免疫原性，和從我國(guó)豐富的花生種質(zhì)資源中篩選低免疫原性或無過敏性的花生品種兩個(gè)方面進(jìn)行了研究。 本文首先確定了花生過敏原粉的制備方法，即經(jīng)過正己烷脫脂后，采用磷酸鹽直接浸提法提取蛋白質(zhì)，超聲波破碎細(xì)胞壁提高蛋白提取率，將離心得到的過敏原液透析，冷凍干燥得過敏原粉，冷凍保存。結(jié)合我國(guó)花生過敏患者血清，初步鑒定了我國(guó)的主要過敏原為Arah1,Arah2和Arah3。 收集了33種中國(guó)花生和23種日本花生，進(jìn)行花生品種間免疫原性差異性比較的研究。利用聚丙烯酰胺凝膠電泳和凝膠成像系統(tǒng)分析了花生的主要過敏原的種類及其含量，采用間接ELISA試驗(yàn)探討了各品種間免疫原性的高低。發(fā)現(xiàn)，綏中大花生和沭陽大站秧缺失Arah3（35，，35kDa），新金伏大、興城大花生、如東碗兒青、日本品種JP-16和JP-19缺失Arah3（24kDa）。56個(gè)品種中Arah1的相對(duì)含量都是最高的，中國(guó)的在25~47.6%范圍內(nèi)，日本的在20.2~39.6%范圍內(nèi)，其次是Arah2（20kDa）和Arah3（14kDa）的相對(duì)含量。通過間接ELISA試驗(yàn)，結(jié)合中國(guó)花生過敏患者的血清，得出中國(guó)花生的免疫原性較日本花生的強(qiáng)。中國(guó)花生品種中免疫原性最低的是20號(hào)鐵嶺四粒紅，最高的是19號(hào)營(yíng)口四粒紅。日本的花生JP-10的免疫原性最強(qiáng)，JP-6的免疫原性最低。花生的免疫原性取決于非常復(fù)雜的因素，單純的過敏條帶的缺失或含量的高低和花生的其免疫原性沒有直接關(guān)系。本試驗(yàn)研究了電子束輻照降低花生過敏原免疫原性的效果及對(duì)其生化性質(zhì)的影響。選擇2、4、 6、8、10、15、20kGy劑量的電子束輻照對(duì)花生過敏原液和脫脂花生粉。輻照后花生主要過敏原（Arah1,Arah2,Arah3）蛋白分子質(zhì)量的變化變化用SDS-聚丙烯酰胺凝膠電泳測(cè)定，免疫原性的變化用免疫印跡和間接競(jìng)爭(zhēng)ELISA法測(cè)定。輻照后花生過敏原溶液的濃度、濁度及疏水性的變化用紫外分光光度計(jì)和熒光光度計(jì)分別進(jìn)行測(cè)定。結(jié)果表明，花生過敏原在溶液狀態(tài)比固體狀態(tài)對(duì)輻照處理更為敏感。當(dāng)輻照劑量低于10kGy時(shí)處理的過敏原液的免疫原性有稍微的增強(qiáng)，劑量大于10kGy時(shí)免疫原性降低，劑量為20kGy時(shí)過敏原液的IC50值是未處理的11倍。輻照使過敏液的濃度和濁度隨著輻照劑量的加大而增加，疏水性與對(duì)照組相比增強(qiáng)，當(dāng)輻照劑量大于15kGy時(shí)又降低。電子束輻照改變了過敏原的生化性質(zhì)，降低了其免疫原性，比較而言，輻照對(duì)液體狀態(tài)下過敏原的影響更顯著。綜上，本文建立了花生過敏原粉制備的方法，初步鑒定了主要過敏原，篩選出缺失過敏原條 帶的品種，證實(shí)了電子束輻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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R155.5
本文編號(hào)：2442669
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R155.5
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