鸡蛋免疫活性ELISA技术及花生致敏蛋白Ara h1亲和纯化技术的研究

发布时间：2018-03-05 02:22

  本文选题：鸡蛋致敏蛋白　切入点：试剂盒　出处：《中国农业科学院》2012年硕士论文　论文类型：学位论文

【摘要】：食物过敏反应是当前世界性的卫生学问题和主要的食品安全问题之一，且随着人们生活水平的提高，食物过敏呈逐年上升的趋势。根据流行病学调查，世界有8%的儿童和2%的成年人对食物过敏，一些发达国家受食物过敏性疾病困扰的人群超过20%。花生和鸡蛋分别被列入8种主要食物过敏原，且其营养丰富而广受消费者的喜爱，而致敏性却严重影响着部分人群的日常生活。因此，研究快速高效的致敏蛋白检测技术和纯化方法显得极其重要。 本课题从鸡蛋致敏蛋白检测方法和花生致敏蛋白纯化方法两方面进行研究，研制高效快速的鸡蛋致敏蛋白ELISA检测试剂盒，实现鸡蛋致敏蛋白的体外快速检测；用免疫亲和柱法纯化花生致敏蛋白Arah1，，实现对致敏蛋白快速高效的分离纯化。 本文的主要研究内容和结果如下：（1）研究并制备了鸡蛋致敏蛋白检测试剂盒。首先通过硫酸铵沉淀法提取鸡蛋中致敏蛋白，用免疫印迹进行活性鉴定，接下来免疫新西兰大白兔制备抗鸡蛋致敏蛋白多克隆抗体。通过对ELISA条件优化，得出碳酸盐缓冲液（pH9.6）为最佳包被缓冲液，先4℃过夜，再37℃放置1h为最佳包被时间，5%的脱脂奶粉为最佳封闭液，酶标二抗的最佳稀释度为2000倍，抗原的最佳反应浓度为1μg/mL，抗体的最佳稀释倍数为50000倍，优化后方法的最低检测限IC10=1.1ng/mL，所建立试剂盒回收率在90%-105%范围之内，特异性良好，试剂盒批内平均变异系数低于5%，批间平均变异系数低于10%，且再现性差异不显著，经37℃加速破坏实验，试剂盒可在4℃可保存6个月，冻融次数不应超过三次，用所建立的方法对样品进行检测分析，检测结果正确率较高。（2）研究了免疫亲合法纯化花生致敏蛋白Arah1。首先通过硫酸铵沉淀法和分子筛层析法提取纯化花生致敏蛋白Arah1，纯度达90%，用所提取的Arah1致敏蛋白免疫新西兰大白兔制备特异性多克隆抗体，效价高达5×105。制备免疫亲和柱：以溴化氰活化的琼脂糖凝胶4B（CBr-activatedSepharose4B）为偶联介质，与抗Arah1特异性多克隆抗体进行偶联（偶联率达到95.9%），制备免疫亲和柱，通过条件优化，筛选出glycine-HCI（pH2.4)为最佳洗脱液，洗脱流速可在0.5～2ml/min内进行选择，具体流速大小可依具体实验而定，选用5min为最佳上样孵育时间，所制备的免疫亲和柱每毫升亲和介质的最高吸附容量在11mg左右，回收率在73.6%-89.2%之间，符合技术要求。综上分析，本研究所制备的鸡蛋致敏蛋白检测试剂盒检测限及稳定性等指标均符合技术要 求，为鸡蛋致敏蛋白的检测分析提供技术支持；对Arah1免疫亲和层析纯化方法的研究表明，此法高效、快速、特异性强，是一种实用性较强的纯化致敏蛋白的方法，为致敏蛋白的纯化及深入研究奠定基础。
[Abstract]:Food allergic reaction is one of the world's major health problems and food safety problems, and with the improvement of people's living standards, food allergies are on the rise year by year. 8% of the world's children and 2% of adults are allergic to food, and more than 20 percent of people in developed countries are suffering from food allergies. Peanuts and eggs are among the eight major food allergens, and they are nutritious and popular among consumers. However, allergenicity seriously affects the daily life of some people. Therefore, it is very important to study the rapid and efficient detection and purification of sensitized proteins. In this paper, the detection method of egg sensitized protein and the purification method of peanut sensitized protein were studied in order to develop an efficient and fast ELISA kit for the detection of egg sensitized protein, so as to realize the rapid detection of egg sensitized protein in vitro. Arabanut sensitized protein Arah1 was purified by immuno-affinity column, and the sensitized protein was separated and purified quickly and efficiently. The main contents and results of this paper are as follows: (1) the detection kit of egg sensitizing protein was studied and prepared. Firstly, the sensitized protein was extracted by ammonium sulfate precipitation method, and the activity was identified by Western blot. Next, New Zealand white rabbits were immunized to prepare polyclonal antibodies against egg sensitized proteins. By optimizing the conditions of ELISA, it was concluded that carbonate buffer (pH 9.6) was the best coating buffer, and spent the night at 4 鈩

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