本文關(guān)鍵詞：蝦（Penaeus Vannamei）過敏原免疫活性的研究　出處：《中國海洋大學(xué)》2006年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 蝦 過敏原 檢測 加工 診斷

【摘要】： 蝦是人類最優(yōu)質(zhì)動物蛋白的來源之一,也是八大類容易引起過敏的食物之一。隨著國際貿(mào)易的發(fā)展,其在食品中的應(yīng)用越來越廣泛,食用蝦引起過敏的潛在危害更值得重視。本論文主要從蝦過敏原蛋白的分離純化、抗體的制備、免疫檢測、不同因子對其免疫活性的影響以及蝦過敏的診斷等方面進行研究,得到如下結(jié)論。 1.不同患者對蝦中過敏原蛋白的識別不同,試驗共識別到8個條帶,其中識別率最高的是分子量為36kD的原肌球蛋白,超過80%的患者血清IgE能夠與其反應(yīng),其次是分子量在85kD的蛋白,除此之外,52kD、41kD、31kD、30kD、22kD的蛋白也都有識別。對不同品種的蝦的免疫識別發(fā)現(xiàn),雖然蝦抽提物的蛋白組分稍有差別,但主要過敏原均為分子量為36kD的原肌球蛋白,其免疫活性沒有明顯的差別。 2.建立了蝦過敏原蛋白活性檢測的Dot-ELISA技術(shù)體系;利用純化的蝦過敏原蛋白作為抗原分別獲得鼠源和兔源的效價為160,000和80,000的抗體。在實驗中,本論文首次采用S180細胞刺激經(jīng)過3次加強免疫的小鼠產(chǎn)生腹水,成功的獲得了效價為40,000的蝦過敏原蛋白抗體,經(jīng)過純化后的抗體效價提高2倍以上,且對蝦過敏原蛋白具有特異性。 3.以獲得的鼠源抗體為一抗,建立了間接競爭酶聯(lián)免疫檢測蝦過敏原蛋白的方法,檢測限為1.85ng/mL,線性檢測范圍為4.79-1400ng/mL,板內(nèi)變異系數(shù)為3.4%～9.1%,板間變異系數(shù)在12.9%～19.6%,檢測限略低于國際同類產(chǎn)品的水平但其他方面均達到或超過國際同類產(chǎn)品的水平。 4.以鼠源抗體為捕獲抗體,膠體金標記的兔源抗體為檢測抗體,制備了蝦過敏原蛋白檢測的免疫滲濾試劑盒,檢測限達到250ng/ml,檢測時間在15min之內(nèi),達到了快速、簡便、特異性的要求,但仍需進一步提高靈敏度。 5.本試驗首次采用超聲波處理蝦過敏原蛋白,結(jié)果表明,在一定的條件下,超聲波能夠降低蝦過敏原蛋白的免疫活性。在0℃的條件下,超聲波處理無論對于蝦抽提物還是蝦肉都沒有明顯的影響。但在50℃條件下,超聲波處理蝦抽提
[Abstract]:Shrimp is one of the best source of animal protein and one of the eight kinds of food which is susceptible to allergies. With the development of international trade, shrimp is more and more widely used in food. The potential harm caused by food shrimp allergy is more worthy of attention. This paper mainly from shrimp allergen protein purification, antibody preparation, immune detection. The effects of different factors on its immune activity and the diagnosis of shrimp allergy were studied. 1. The recognition of allergen proteins in different patients was different. Eight bands were identified in the experiment, in which the highest recognition rate was 36kD promyosin. More than 80% of the patients could react with IgE, followed by protein with molecular weight of 85kD, in addition to 52kD 41kDX 31kDN 30kD. The protein of 22kD was also recognized. The immunological recognition of different species of shrimp showed that, although the protein components of the extract were slightly different, the main allergens were all promyosin with molecular weight of 36kD. There was no significant difference in immune activity. 2. The Dot-ELISA technique system for detecting the activity of shrimp allergen protein was established. The purified shrimp allergen protein was used as antigen to obtain antibodies with titers of 160,000 and 80,000 from mouse and rabbit, respectively. In this paper, we first used S180 cells to stimulate mice to produce ascites after three times of booster immunization, and successfully obtained the antibody of shrimp allergen protein with titer of 40,000. After purification, the titer of the antibody was increased by more than 2 times, and the allergen protein of prawn was specific. 3. An indirect competitive enzyme-linked immunosorbent assay (Elisa) was developed for the detection of shrimp allergen proteins with a detection limit of 1.85 ng / mL. The linear detection range was 4.79-1400ng / mL, the coefficient of variation was 3.4% and the coefficient of variation between plates was 12.9ng / mL. The detection limit is slightly lower than the international level of similar products, but other aspects are up to or above the level of international products of the same kind. 4. Using mouse antibody as capture antibody and colloidal gold labeled rabbit antibody as detection antibody, an immunofiltration kit for shrimp allergen protein detection was prepared. The detection limit was 250 ng / ml. The detection time was within 15 min, which met the requirements of rapid, simple and specific, but the sensitivity still needed to be improved. 5. The results showed that ultrasound could reduce the immunological activity of shrimp allergen protein under certain conditions, and at 0 鈩，

本文編號：1419946