杨梅主要过敏原分离纯化以及免疫活性鉴定

发布时间：2018-03-05 20:16

本文选题：杨梅　切入点：过敏原　出处：《浙江大学》2015年硕士论文　论文类型：学位论文

【摘要】：对于大多数人来讲,水果是一种健康食品,但有一部分人由于自身遗传以及生活环境等因素,对某一种或者多种水果过敏。这种现象伴随水果生产专业化以及销售网络全球化以及消费水果种类和数量的增加而更为普遍。水果过敏是患者的免疫系统发生紊乱而导致的,它主要是由于某些蛋白成分(过敏原)所引起的,一般属于Ⅰ型超敏反应(type I hypersensitivity),约1-3%的人群受到此类型疾病所困扰。因此,开展水果的过敏原研究对保护人们的生命健康、提高人民生活质量具有巨大的经济效益以及社会效益。本论文主要是从杨梅过敏原蛋白的分离纯化以及纯化产物的免疫学活性分析等方面进行研究,得到如下结论：首先,应用Coca's提取法提取杨梅果实中粗蛋白液,运用还原SDS-PAGE技术对杨梅粗提蛋白液进行初步分析,显示杨梅粗提蛋白中包含以前用过敏血清免疫印迹实验鉴定为28kDa的主要过敏原蛋白条带。其次,通过超滤离心法、SP-FF离子交换层析以及Superdex7510/300GL凝胶柱层析方法分别纯化杨梅主要过敏蛋白。依据杨梅转录组推导的氨基酸数据,对杨梅主要过敏蛋白进行肽段指纹图谱、分子量质谱测定以及酶联免疫(ELISA)间接法分析与鉴定,得到杨梅主要过敏原理论分子量和质谱检测所得分子量一致,ELISA检测后有很强的免疫活性,且肽段指纹图谱匹配实验与氨基酸序列比对得出杨梅主要过敏蛋白属于几丁质酶第三家族(class Ⅲ chitinase)(已提交至NCBI数据库,序列号：KP307670)。本研究初步对杨梅主要过敏原进行分离纯化以及免疫活性的鉴定分析,可在ELISA检测中作为杨梅特异性过敏原,为杨梅过敏反应疾病的诊断奠定了良好的基础。
[Abstract]:For most people, fruit is a healthy food, but some people because of their own inheritance and living environment and other factors, Allergies to one or more fruits are more common with specialization in fruit production, globalization of distribution networks, and an increase in the variety and quantity of fruit consumed. Fruit allergies are the result of a disorder in the patient's immune system. It is mainly caused by certain protein components (allergens), which generally belong to type I hypersensitivity, and about 1 to 3% of the population are affected by this type of disease. Therefore, the study of allergen in fruits can protect people's life and health. Improving people's quality of life has great economic and social benefits. In this paper, the purification of myricetin and the analysis of immunological activity of the purified product are studied, and the conclusions are as follows:. Firstly, the crude protein solution was extracted from the fruit of Myrica rubra by Coca's extraction method, and the crude protein extract of Myrica rubra was preliminarily analyzed by reducing SDS-PAGE technique. The results showed that the crude extract protein of Myrica rubra contained the main allergen protein bands identified as 28kDa by the immunoblotting assay of anaphylaxis serum. Secondly, the main allergic proteins of Myrica rubra were purified by ultrafiltration centrifugation with SP-FF ion exchange chromatography and Superdex7510/300GL gel column chromatography. According to the amino acid data derived from the transcriptome of Myrica myrica, the peptide fingerprint of the main allergic proteins of Myrica rubra was carried out. Molecular weight mass spectrometry and Elisa indirect analysis and identification showed that the theoretical molecular weight of the main allergen of Myrica rubra and the molecular weight of the main allergen detected by mass spectrometry were consistent with those of Elisa, and that the molecular weight of the main allergen of Myrica rubra had strong immunological activity. The peptide fingerprint matching experiment and amino acid sequence alignment showed that the main allergic protein of Myrica rubra belongs to the third family of chitinase (submitted to NCBI database, serial number: KP307670). In this study, the main allergens of Myrica rubra were isolated and purified and their immunological activities were identified and analyzed, which can be used as a specific allergen in the detection of Myrica rubra by ELISA, which lays a good foundation for the diagnosis of myrica anaphylaxis disease.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R446.6

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