虾（Penaeus Vannamei）过敏原免疫活性的研究

发布时间：2018-01-13 18:08

  本文关键词：虾（Penaeus Vannamei）过敏原免疫活性的研究　出处：《中国海洋大学》2006年博士论文　论文类型：学位论文

  更多相关文章： 虾 过敏原 检测 加工 诊断

【摘要】： 虾是人类最优质动物蛋白的来源之一,也是八大类容易引起过敏的食物之一。随着国际贸易的发展,其在食品中的应用越来越广泛,食用虾引起过敏的潜在危害更值得重视。本论文主要从虾过敏原蛋白的分离纯化、抗体的制备、免疫检测、不同因子对其免疫活性的影响以及虾过敏的诊断等方面进行研究,得到如下结论。 1.不同患者对虾中过敏原蛋白的识别不同,试验共识别到8个条带,其中识别率最高的是分子量为36kD的原肌球蛋白,超过80%的患者血清IgE能够与其反应,其次是分子量在85kD的蛋白,除此之外,52kD、41kD、31kD、30kD、22kD的蛋白也都有识别。对不同品种的虾的免疫识别发现,虽然虾抽提物的蛋白组分稍有差别,但主要过敏原均为分子量为36kD的原肌球蛋白,其免疫活性没有明显的差别。 2.建立了虾过敏原蛋白活性检测的Dot-ELISA技术体系;利用纯化的虾过敏原蛋白作为抗原分别获得鼠源和兔源的效价为160,000和80,000的抗体。在实验中,本论文首次采用S180细胞刺激经过3次加强免疫的小鼠产生腹水,成功的获得了效价为40,000的虾过敏原蛋白抗体,经过纯化后的抗体效价提高2倍以上,且对虾过敏原蛋白具有特异性。 3.以获得的鼠源抗体为一抗,建立了间接竞争酶联免疫检测虾过敏原蛋白的方法,检测限为1.85ng/mL,线性检测范围为4.79-1400ng/mL,板内变异系数为3.4%～9.1%,板间变异系数在12.9%～19.6%,检测限略低于国际同类产品的水平但其他方面均达到或超过国际同类产品的水平。 4.以鼠源抗体为捕获抗体,胶体金标记的兔源抗体为检测抗体,制备了虾过敏原蛋白检测的免疫渗滤试剂盒,检测限达到250ng/ml,检测时间在15min之内,达到了快速、简便、特异性的要求,但仍需进一步提高灵敏度。 5.本试验首次采用超声波处理虾过敏原蛋白,结果表明,在一定的条件下,超声波能够降低虾过敏原蛋白的免疫活性。在0℃的条件下,超声波处理无论对于虾抽提物还是虾肉都没有明显的影响。但在50℃条件下,超声波处理虾抽提
[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，

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