章鱼过敏原的研究

发布时间：2018-06-05 12:45

本文选题：章鱼 + 过敏原　；参考：《集美大学》2012年硕士论文

【摘要】：食物过敏是人类比较常见的一种过敏性疾病，主要是由食物中的过敏原引起。人们发生食物过敏后，常出现腹泻、哮喘、呕吐等症状，严重时会虚脱、休克，甚至危及生命。水产类食物作为重要的过敏食物之一，由其引起的过敏反应引起了世界人们的关注，在联合国粮农组织公布的八大类过敏食物中，水产品就占了两大类。章鱼作为重要的水产资源，因其肉质鲜嫩、味道鲜美，具有很高的营养价值，深受消费者喜爱。随着章鱼消费量的增加，有关章鱼过敏的报道也随之增多，但是关于章鱼过敏原的研究较少，集中于主要过敏原—原肌球蛋白（TM）的纯化及分析，对于章鱼是否含有其他过敏原以及如何开发低致敏性章鱼产品等方面尚未见报道。本实验首先对章鱼TM进行分离纯化、鉴定及性质分析，并制备多克隆动物抗体；然后对章鱼TM分别进行超声联合加热、紫外照射、高温高压和美拉德反应等加工处理方式；并采用模拟胃肠液消化实验对不同处理后TM的消化稳定性进行分析，进一步运用水产食物过敏患者血清对其致敏性进行鉴定；最后，对章鱼肌肉进行热处理和高温高压处理，分析肌肉蛋白和TM的性质变化。结果显示，纯化章鱼TM的分子量为35kDa，与蟹类TM具有免疫交叉反应，是含糖量为1.3%的糖蛋白，且对热和酸碱稳定；超声联合加热和紫外照射对章鱼TM消化稳定性及免疫活性影响不明显，高温高压处理能够有效降低TM的消化稳定性和免疫活性；章鱼TM与葡萄糖发生美拉德反应，随着反应时间的延长，其产物的免疫活性逐渐降低，但其消化稳定性却有所增加；对章鱼肌肉进行100℃蒸煮10min和高温高压10min处理，发现两种处理方式均能够一定程度地降低章鱼TM的消化稳定性，且高温高压处理效果更显著。本实验还对章鱼精氨酸激酶（AK）进行分离纯化及鉴定，制备多克隆抗体；然后对其致敏性、热稳定性、pH稳定性及消化稳定性等理化性质进行分析；最后将其与甲壳类动物过敏原AK进行分析比较。结果显示，采用水产食物过敏患者血清对章鱼肌浆蛋白进行免疫反应，发现能与血清发生特异性反应的有50kDa、38kDa和26kDa蛋白组分，其中最主要的是38kDa蛋白组分；进一步纯化得到该38kDa蛋白组分，应用肽指纹图谱对其进行分析鉴定，确定为章鱼AK；对章鱼AK的致敏性进行分析，确定其为章鱼的一种新型过敏原，并制备得到了效价较高的多克隆动物抗体；章鱼AK是含糖量为1.1%的糖蛋白，且对热和酸碱均不稳定，经过不同条件的热处理和pH处理后，章鱼AK的免疫活性均受到明显影响；章鱼AK容易被胃蛋白酶、胰蛋白酶及胰凝乳蛋白酶等消化酶降解。对章鱼AK进行分子克隆，得到其全长基因序列和氨基酸序列，并将章鱼AK与甲壳类动物过敏原AK进行比较分析。结果显示，章鱼AK氨基酸序列比甲壳类动物AK少8个氨基酸残基，且二者序列相似性仅为54%，但二者蛋白三维结构高度相似，且抗原表位预测结果同样具有一定的相似性；章鱼AK能和甲壳类动物AK的多克隆动物抗体发生明显的免疫交叉反应；此外，章鱼AK与甲壳类动物AK具有相似的致敏性，蟹AK能够抑制章鱼AK与IgE之间的特异性反应。总之，本研究主要是对章鱼的过敏原TM和AK进行了详细分析。首先是对章鱼的主要过敏原TM进行模拟加工处理，以期筛选出能够有效降低章鱼TM消化稳定性和免疫活性的加工方法；其次是首次证明了AK是章鱼的一种新型过敏原，，并对其理化性质进行进一步分析，同时将其与虾蟹过敏原AK进行比较，分析二者之间的相似性。这些结果可望为开发低致敏性章鱼产品提供技术参考，为章鱼过敏的诊断和治疗提供理论依据。
[Abstract]:Food allergy is one of the most common allergic diseases in human beings, mainly caused by allergen in food. After food allergy, people often suffer from symptoms of diarrhea, asthma, vomiting and other symptoms, such as deactivation, shock, and even life-threatening. Aquatic food is one of the most important allergic foods, and the allergic reaction caused by it causes the world. Among the eight categories of allergic food published by the United Nations Food and Agriculture Organization, the aquatic products account for two major categories. Octopus, as an important aquatic resource, has high nutritional value because of its delicate meat quality and delicious taste, and is very popular with consumers. With the increase of Octopus consumption, reports on Octopus allergies have increased, but the increase in Octopus allergies is increasing. There are few studies on the allergens of octopus, which are concentrated on the purification and analysis of the main allergens - promyosin (TM). There are no reports on whether Octopus contains other allergens and how to develop low sensitizing Octopus products.
In this experiment, the TM of Octopus was isolated, identified and analyzed, and the polyclonal antibody was prepared. Then the octopus TM was processed by ultrasonic combined heating, ultraviolet radiation, high temperature and high pressure and Maillard reaction, and the digestive stability of TM after different treatments was divided by simulated gastrointestinal digestion experiment. Furthermore, the sensitivities of the patients with aquaculture food allergy were further identified. Finally, the changes in the properties of muscle protein and TM were analyzed by heat treatment and high temperature and pressure treatment of Octopus muscles. The results showed that the molecular weight of the purified octopus TM was 35kDa, and it had a immune cross reaction with the crab TM, and the sugar content was 1.3%. Heat and acid and alkali are stable; ultrasonic combined heating and ultraviolet irradiation have no obvious effects on the digestive stability and immune activity of octopus TM. High temperature and high pressure treatment can effectively reduce the digestive stability and immune activity of TM. The TM and glucose of Octopus have mallard reaction. With the prolonged reaction time, the immune activity of the products gradually decreases, but the effect of the reaction is gradually reduced. The digestive stability was increased, and 100 centigrade steaming 10min and high temperature and high pressure 10min treatment of Octopus muscle found that the two treatments could reduce the digestive stability of the octopus TM to a certain extent, and the effect of high temperature and high pressure treatment was more significant.
The isolation, purification and identification of Octopus arginine kinase (AK), the preparation of polyclonal antibody, and the analysis of its physicochemical properties, such as sensitization, thermal stability, pH stability and digestive stability, were analyzed in the experiment. The results were compared with the allergen AK of the crustaceans. The results showed that the serum of patients with aquatic food allergy was used. The immunoreaction of Octopus sarcoplasmic protein (Octopus sarcoplasmic protein) showed that the protein components of 50kDa, 38kDa and 26kDa could be specific to the serum. The most important component was the 38kDa protein component; the 38kDa protein component was further purified, and the peptide fingerprint was used to identify and identify the octopus AK, and the sensitization of the octopus AK was analyzed. A new type of allergens of Octopus was determined and the polyclonal antibody of high titer was obtained. Octopus AK was a glycoprotein containing 1.1% sugar, and both heat and acid and alkali were not stable. After different conditions of heat treatment and pH treatment, the immunological activity of octopus AK was obviously affected; octopus AK was easy to be pepsin, pancreatic eggs. The molecular cloning of octopus AK was cloned and its full length gene sequence and amino acid sequence were obtained, and the AK of octopus and the allergens of crustaceans were compared and analyzed. The results showed that the AK amino acid sequence of octopus AK was less than 8 amino acid residues in the crustaceans, and the sequence similarity of the two was only 54%. However, the three-dimensional structure of the two proteins is highly similar, and the prediction results of antigen epitopes are similar. Octopus AK can have obvious immunological cross reaction with the polyclonal antibody of crustacean AK; in addition, the octopus AK has similar sensitivities to the crustacean AK, and the crab AK can inhibit the specificity between the octopus AK and IgE. Sexual reaction.
In conclusion, this study is mainly to analyze the allergens of octopus TM and AK in detail. First, the main allergen TM of octopus is simulated and processed in order to screen out the processing methods that can effectively reduce the digestive stability and immune activity of octopus TM. Secondly, it is the first time that AK is a new type of allergens of octopus. Further analysis was carried out, and compared with the allergen AK of shrimp and crab, the similarity between the two was analyzed. These results may provide a technical reference for the development of low sensitizing Octopus products and provide a theoretical basis for the diagnosis and treatment of Octopus allergies.
【学位授予单位】：集美大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：S917.4;R392

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