虾类过敏原的活性分析及其抗原表位的研究

发布时间：2018-07-11 13:56

本文选题：虾过敏原 + 鉴定　；参考：《中国海洋大学》2011年硕士论文

【摘要】：随着现代社会的发展,人们饮食结构的变化,与食物直接相关的过敏疾病发病率日益增高,给人们的生命健康造成了巨大威胁。虾类产品属于我国产值比较高的甲壳类海产品,由于味道鲜美、营养丰富,深受消费者的喜爱。然而虾类海产品属于8大类食物过敏原之一,使许多消费者饱受过敏症状的困扰。本论文着重对虾类过敏原的鉴定和活性消除方法进行研究,结合生物信息学方法进一步分析过敏原的表位区域及氨基酸组成性质,并对表位进行空间定位。主要内容如下: 1、以刀额新对虾为研究对象,分离和纯化了分子量为36kD的主要过敏原蛋白,利用基质辅助激光解析电离/飞行时间质谱对其进行肽质量指纹图谱鉴定,并对得到的结果采用Mascot搜索引擎在NCBInr数据库上进行搜索。结果表明刀额新对虾主要过敏原为原肌球蛋白,与斑节对虾原肌球蛋白匹配分值最高为268,吻合肽段27条,序列覆盖率为65%;与其它无脊椎动物如腐食酪螨、衣鱼等的原肌球蛋白的序列覆盖率也很高。这一结果不仅表明了刀额新对虾与其它甲壳类海产品过敏原存在着高度同源性,而且为其与甲壳类及其它无脊椎动物主要过敏原之间存在严重交叉反应的现象提供了理论依据。 2、在鉴定了刀额新对虾的主要过敏原之后,进一步分析了常见热加工方式对其过敏原活性的影响。选取煮、蒸、高压这三种方法,分别处理刀额新对虾不同时间。通过检测总蛋白和主要过敏原含量以及活性的变化,分析对虾类过敏原的影响。结果表明经过三种热处理后,36kD主要过敏原蛋白仍然存在,但其活性有不同程度的下降。在25-35kD区域出现一条新的免疫活性蛋白,但影响不大,全蛋白的免疫活性仍有不同程度的降低。其中高压对活性的降低程度最大,30分钟时免疫活性降低了97%。但质地剖面分析结果表明,高压处理对虾肉质构破坏最大,仍需进一步优化高压处理工艺,在降低虾致敏活性的同时,保持其口感及营养。 3、利用生物信息学方法对虾过敏原及其他甲壳类过敏原之间的同源性进行分析,并绘制出系统进化树。结果表明甲壳类过敏原原肌球蛋白氨基酸序列十分保守,序列之间同源性很高。其中斑节对虾、凡纳滨对虾过敏原同褐美对虾主要过敏原Pen a 1的氨基酸序列完全相同。采用DNAstar、AntheProt等生物信息学软件分析Pen a 1的二级结构、亲水性、溶剂可及性、可塑性、抗原指数等多个性质,间接预测线性表位区域,并结合2个在线网站对表位的预测结果进行筛选,得到10个抗原表位后进行表位肽的固相合成。采用竞争Dot-blot方法,利用患者血清对表位肽的活性进行初步验证。结果表明其中8个表位多肽为主要过敏原表位,其中有两个以前未曾有研究者报道过。对表位区域的氨基酸分析中发现精氨酸(R)、酪氨酸(Y)、苯丙氨酸(F)、丝氨酸(S)以及谷氨酸(E)这5种氨基酸在表位区域出现概率很高。 4、以猪原肌球蛋白1c1gC为模板,利用SWISS-MODEL以及CPHmodels中相关的同源建模功能对Pen a 1的空间结构进行预测。采用原子平均势能、分子体系动力学分析以及拉氏构象图对建模结果进行评估。结果表明模拟形成的Pen a 1构象有较高的稳定性和合理性。从构象中可以看出, Pen a 1空间结构比较简单,没有复杂的三、四级结构,二级结构主要以α-螺旋为主。从构象方面分析,Pen a 1单链自身形成构象型表位的几率不大,而线性表位在空间中的定位结果也显示基本上线性表位全部暴露在外。 5、利用氨基酸突变的方法研究Pen a 1表位中的关键氨基酸,选择Pen a 1中peptide6和peptide10这两条表位肽,将其中的活性氨基酸替代为人或猪原肌球蛋白中相应的非活性氨基酸。采用Dot-blot竞争酶联免疫的方法检测突变表位肽过敏活性的变化,分析对活性影响较大的氨基酸残基。结果表明,Pen a 1中Peptide 10中所对应的表位区域中,278F和279S氨基酸替代为人原肌球蛋白相应位置的氨基酸L后,突变表位肽与过敏患者血清IgE的结合能力有明显的下降,说明这两者为该表位中的关键氨基酸。其他表位中的关键氨基酸还有待于进一步分析。
[Abstract]:With the development of modern society, the change of people's diet structure, the incidence of allergic diseases which are directly related to food is increasing, which poses a great threat to people's life and health. Shrimp products belong to the high value crustaceans of our country, which are delicious, rich in camping and are loved by consumers. However, shrimp seafood is a kind of seafood. It is one of the 8 major food allergen, which makes many consumers plagued by allergy symptoms. This paper focuses on the identification and activity elimination methods of shrimp allergen, and further analyzes the surface and amino acid composition of allergens with bioinformatics methods, and makes spatial location of the epitopes. The main contents are as follows:
1, the main allergen protein with molecular weight of 36kD was isolated and purified, and the peptide mass fingerprint was identified by matrix assisted laser analytical ionization / time of flight mass spectrometry, and the results were searched by the Mascot search engine on the NCBInr database. The results showed that the knife forehead was the new prawn owner. The anaphylaxis was original myosin, and the maximum matching score of the myosin of Penaeus Penaeus was 268, the peptide segment was 27, the sequence coverage was 65%, and the sequence coverage of the myosin of other invertebrates, such as the chitin mite and the coat fish, was also high. This result not only indicated the allergens of the new shrimp and other crustaceans in the shrimp and other crustaceans. There is a high degree of homology and provides a theoretical basis for the severe cross reaction between them and crustaceans and other invertebrates.
2, after identifying the main allergen of Penaeus Penaeus, the influence of the common hot processing methods on the activity of allergens was further analyzed. The three methods of cooking, steaming and high pressure were selected to deal with the different time of the new prawns. The changes of the total protein, the main allergens and the activity of the allergens were detected, and the shadow of the shrimp allergen was analyzed. The results showed that after three kinds of heat treatment, the main allergen protein of 36kD still existed, but its activity decreased in varying degrees. There was a new immuno active protein in the 25-35kD region, but the effect of the whole protein was still reduced in varying degrees. Among them, the activity was most reduced by high pressure, and the immunity was 30 minutes. The activity of 97%. was reduced, but the texture profile analysis showed that high pressure treatment of shrimp meat texture was the greatest damage. It still needed to further optimize the high pressure treatment process, while reducing the sensitization activity of shrimp, maintaining its taste and nutrition.
3, using bioinformatics method to analyze the homology of shrimp allergen and other crustacean allergens, and draw a phylogenetic tree. The results show that the amino acid sequence of the crustacean allerogen myosin amino acid is very conservative and the sequence is very homologous. The amino acid sequence of the Pen a 1 is exactly the same.
DNAstar, AntheProt and other bioinformatics software are used to analyze the two grade structure of Pen a 1, hydrophilicity, solvent accessibility, plasticity and antigen index, and indirectly predict the linear epitope region, and combine 2 online sites to screen the prediction results of the epitopes and obtain the solid phase synthesis of epitopes after 10 epitopes. The Dot-blot method was used to verify the activity of the epitope of the patient's sera. The results showed that 8 epitopes were the main allergenic epitopes, and two of them had not been reported before. The amino acid analysis of the epitopes found that arginine (R), tyrosine (Y), phenylalanine (F), serine (S), and glutamic acid (E) were found. The probability of the 5 amino acids in the epitope region is very high.
4, using the porcine proomyosin 1c1gC as a template, the spatial structure of Pen a 1 was predicted by using the related homologous modeling functions of SWISS-MODEL and CPHmodels. The results were evaluated by atomic mean potential energy, molecular dynamics analysis and Lagrangian conformation diagram. The results showed that the conformation of the Pen a 1 formed by the simulation had high stability. It can be seen from the conformation that the spatial structure of Pen a 1 is simple, there is no complex three, four structure, and the two structure is mainly alpha helix. From the conformation analysis, the probability of forming conformational epitopes of the single strand of Pen a 1 is not good, and the linear epitope in the space also shows the basic linear epitopes. Exposure to the outside.
5, the key amino acids in the Pen a 1 epitopes were studied by amino acid mutation, and two epitopes of peptide6 and peptide10 in Pen a 1 were selected to substitute the active amino acids in the human or porcine promyosin as the corresponding inactive amino acids. The mutation of the mutant epitopes was detected by the Dot-blot competitive enzyme immunoassay. The results showed that in the epitope region corresponding to the Peptide 10 in Pen a 1, 278F and 279S amino acids were substituted for the corresponding amino acid L of human myosin, and the binding ability of the mutant epitopes to the serum IgE in the allergic patients was significantly decreased, indicating that both of these were the key points in the epitope. Key amino acids. Other key amino acids in other epitopes need further analysis.
【学位授予单位】：中国海洋大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

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