本文選題：雞蛋致敏蛋白　切入點：試劑盒　出處：《中國農(nóng)業(yè)科學院》2012年碩士論文　論文類型：學位論文

【摘要】：食物過敏反應(yīng)是當前世界性的衛(wèi)生學問題和主要的食品安全問題之一，且隨著人們生活水平的提高，食物過敏呈逐年上升的趨勢。根據(jù)流行病學調(diào)查，世界有8%的兒童和2%的成年人對食物過敏，一些發(fā)達國家受食物過敏性疾病困擾的人群超過20%�；ㄉ碗u蛋分別被列入8種主要食物過敏原，且其營養(yǎng)豐富而廣受消費者的喜愛，而致敏性卻嚴重影響著部分人群的日常生活。因此，研究快速高效的致敏蛋白檢測技術(shù)和純化方法顯得極其重要。 本課題從雞蛋致敏蛋白檢測方法和花生致敏蛋白純化方法兩方面進行研究，研制高效快速的雞蛋致敏蛋白ELISA檢測試劑盒，實現(xiàn)雞蛋致敏蛋白的體外快速檢測；用免疫親和柱法純化花生致敏蛋白Arah1，，實現(xiàn)對致敏蛋白快速高效的分離純化。 本文的主要研究內(nèi)容和結(jié)果如下：（1）研究并制備了雞蛋致敏蛋白檢測試劑盒。首先通過硫酸銨沉淀法提取雞蛋中致敏蛋白，用免疫印跡進行活性鑒定，接下來免疫新西蘭大白兔制備抗雞蛋致敏蛋白多克隆抗體。通過對ELISA條件優(yōu)化，得出碳酸鹽緩沖液（pH9.6）為最佳包被緩沖液，先4℃過夜，再37℃放置1h為最佳包被時間，5%的脫脂奶粉為最佳封閉液，酶標二抗的最佳稀釋度為2000倍，抗原的最佳反應(yīng)濃度為1μg/mL，抗體的最佳稀釋倍數(shù)為50000倍，優(yōu)化后方法的最低檢測限IC10=1.1ng/mL，所建立試劑盒回收率在90%-105%范圍之內(nèi)，特異性良好，試劑盒批內(nèi)平均變異系數(shù)低于5%，批間平均變異系數(shù)低于10%，且再現(xiàn)性差異不顯著，經(jīng)37℃加速破壞實驗，試劑盒可在4℃可保存6個月，凍融次數(shù)不應(yīng)超過三次，用所建立的方法對樣品進行檢測分析，檢測結(jié)果正確率較高。（2）研究了免疫親合法純化花生致敏蛋白Arah1。首先通過硫酸銨沉淀法和分子篩層析法提取純化花生致敏蛋白Arah1，純度達90%，用所提取的Arah1致敏蛋白免疫新西蘭大白兔制備特異性多克隆抗體，效價高達5×105。制備免疫親和柱：以溴化氰活化的瓊脂糖凝膠4B（CBr-activatedSepharose4B）為偶聯(lián)介質(zhì)，與抗Arah1特異性多克隆抗體進行偶聯(lián)（偶聯(lián)率達到95.9%），制備免疫親和柱，通過條件優(yōu)化，篩選出glycine-HCI（pH2.4)為最佳洗脫液，洗脫流速可在0.5～2ml/min內(nèi)進行選擇，具體流速大小可依具體實驗而定，選用5min為最佳上樣孵育時間，所制備的免疫親和柱每毫升親和介質(zhì)的最高吸附容量在11mg左右，回收率在73.6%-89.2%之間，符合技術(shù)要求。綜上分析，本研究所制備的雞蛋致敏蛋白檢測試劑盒檢測限及穩(wěn)定性等指標均符合技術(shù)要 求，為雞蛋致敏蛋白的檢測分析提供技術(shù)支持；對Arah1免疫親和層析純化方法的研究表明，此法高效、快速、特異性強，是一種實用性較強的純化致敏蛋白的方法，為致敏蛋白的純化及深入研究奠定基礎(chǔ)。
[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

本文編號：1568344