刀额新对虾主要致敏原的分离纯化与鉴定

发布时间：2018-04-15 21:19

本文选题：刀额新对虾 + 过敏原　；参考：《江南大学》2008年硕士论文

【摘要】： 食物过敏作为一类食源性疾病已引起了人们的普遍关注。过敏性疾病已被世界卫生组织列为二十一世纪重点防沫的三大类疾病之一,是当前世界性的重大卫生学问题。食物过敏作为一类常见的过敏性疾病,引起的临床症状主要有头晕、头痛、胸闷、恶心、呕吐、皮肤痉痒等,严重时还会休克甚至死亡。因此,开展食物(特别是海产品)的过敏原研究对保护人们生命健康,提高人民生活质量具有巨大的经济效益和社会效益。虾蟹等甲壳类动物及其制品是人类优质动物蛋白的来源之一,也是八大类容易引起过敏的食物之一。随着国际贸易的发展,其在食品中的应用越来越广泛,食用虾引起过敏的潜在危害更值得重视。本论文选用我国华南地区常见的食用虾种,刀额新对虾(Metapenaeus ensis俗名基围虾),作为研究对象,主要从虾过敏原蛋白的分离纯化、纯化产物的免疫学鉴定等方面进行研究,得到如下结论: 首先,应用SDS-PAGE技术对粗提蛋白液进行初步分离,SDS-PAGE图谱显示刀额新对虾粗提蛋白约有20条可辨蛋白带,分子量在12.1 KDa～105.6 KDa之间。利用12例虾过敏患者血清通过免疫印迹(western-blotting)分析,初步鉴定出刀额新对虾粗提液中有7条过敏蛋白条带,其分子量分别为36 KDa、46.2 KDa、60 KDa、64.8 KDa、68 KDa、78 KDa和88.5 KDa。另外还有21.6 KDa的蛋白也被个别患者的血清识别,其中4条过敏蛋白条带反应率高,为主要致敏组分,分子量分别是36 KDa、46.2KDa、60 KDa和68 KDa,这与国内外其他人的研究结果有一定差异,这可能与虾的种类不同、各地区虾类生长环境的不一致性以及过敏患者人群的个体差异有关。其次,通过硫酸铵分段盐析、DEAE-52离子交换层析和Sephadex G-100凝胶柱层析等方法逐步纯化了虾过敏蛋白。并通过免疫印迹(western-blotting)、斑点酶联免疫(dot-ELISA)、和酶联免疫(ELISA)间接法对已经纯化的虾过敏蛋白进行了分析和鉴定。具有很高的反应性。本研究初步纯化、鉴定出刀额新对虾主要过敏原蛋白,为进一步研究虾类过敏原的结构与功能奠定了一定的理论基础,其在ELISA检测中作为特异性抗原与虾过敏症患者血清中特异性IgE有良好的反应性,也为虾类食物过敏反应疾病的诊断和进一步研制脱敏试剂奠定了良好的基础。
[Abstract]:Food allergy, as a kind of foodborne disease, has attracted people's attention.Allergic diseases have been listed by the World Health Organization as one of the three major anti-foaming diseases in the 21 century.Food allergy as a common allergic disease, the main clinical symptoms caused by dizziness, headache, chest tightness, nausea, vomiting, skin spasmolysis and itching, severe shock and even death.Therefore, the development of food (especially seafood) allergen research to protect people's life and health, improve people's quality of life has great economic and social benefits.Crustaceans such as shrimps and crabs and their products are one of the sources of human high quality animal protein and one of the eight kinds of food which can easily cause allergies.With the development of international trade, its application in food is more and more extensive.In this paper, the common edible shrimp species in South China, Metapenaeus ensis, as the research object, were studied from the isolation and purification of the allergen protein and the immunological identification of the purified products.The following conclusions have been drawn:Firstly, the SDS-PAGE analysis of crude protein extract by SDS-PAGE showed that there were about 20 discernible protein bands with molecular weight between 12.1 KDa~105.6 KDa and 12.1 KDa~105.6 KDa.Using Western blotting analysis of the sera of 12 shrimp allergy patients, we preliminarily identified 7 bands of allergy protein in the crude extract of Penaeus Monodon, the molecular weight of which was 36 K Dax 46.2 K Da1 60 K Da4. 8 K DaDX 68 KDaf78 KDa and 88 5 K Da. respectively.In addition, the protein of 21.6 KDa was also recognized by the serum of individual patients. The reaction rate of four allergic protein bands was high, and the molecular weight was 36KDa46.2KDa-60 KDa and 68kDa. the results were different from those of other people at home and abroad.This may be related to the different species of shrimp, the inconsistency of shrimp growth environment in different regions and the individual difference of allergy patients.Secondly, shrimp allergy protein was purified by ammonium sulfate fractionated DEAE-52 ion exchange chromatography and Sephadex G-100 gel column chromatography.The purified shrimp allergy proteins were analyzed and identified by Western blotting, dot-ELISAA and Elisa indirect methods.Be highly reactive.In this study, the main allergen proteins were identified by purification, which laid a theoretical foundation for further study on the structure and function of shrimp allergens.As a specific antigen in the detection of ELISA, it has good reactivity with specific IgE in the sera of shrimp hypersensitivity patients. It also lays a good foundation for the diagnosis of shrimp food allergy disease and the further development of desensitizing reagent.
【学位授予单位】：江南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

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