鸡蛋蛋清溶菌酶分离纯化及其抗原性评估

发布时间：2018-05-19 01:16

  本文选题：溶菌酶 + 分离纯化　； 参考：《南昌大学》2010年硕士论文

【摘要】： 鸡蛋溶菌酶是一种天然存在的具有杀菌抑菌作用的碱性蛋白酶,在食品行业中得到了广泛的应用,然而作为鸡蛋中的一种主要过敏原,约有35%的鸡蛋过敏患者对溶菌酶过敏。另外,食物过敏是一种复杂的网络免疫失调症,多种抗原抗体之间相互作用,探明各种过敏原在食物过敏反应中的相互关系,对于低过敏或无过敏食品的开发具有重要的指导意义。因此,本论文分离纯化高纯度鸡蛋溶菌酶,并对其抗原性进行初步评估,这不仅为溶菌酶的工业化生产提供一定的技术支持,还能够为低过敏性或无过敏性蛋制品的开发提供一些基本的实验参数。 本研究的主要内容包括鸡蛋溶菌酶的分离纯化、溶菌酶酶活性质研究、兔抗溶菌酶多克隆抗体的制备以及溶菌酶抗原性评估。 在鸡蛋溶菌酶分离纯化过程中,建立了CM-Sepharose Fast Flow离子交换层析分离溶菌酶的方法。分离得到的样品经5%-15%SDS-PAGE电泳进行鉴定,结果表明,溶菌酶纯度在90%以上,回收率在31.1%左右。 在溶菌酶酶活性质研究过程中,以枯草芽孢杆菌及其细胞壁作为研究对象,研究了鸡蛋溶菌酶的部分酶活性质。研究结果表明,鸡蛋溶菌酶对枯草芽孢杆菌的生长具有明显的抑制作用,在50℃环境中,溶菌酶对枯草芽孢杆菌细胞壁具有明显的降解作用,但是在25℃时的作用并不明显,这说明温度对溶菌酶酶活有很大影响。 在兔抗溶菌酶多克隆抗体制备过程中,通过弗氏佐剂来加强免疫,采用皮下多点免疫的方法免疫日本大白兔,分离血清,然后用间接ELISA检测了兔抗溶菌酶血清效价的变化。在测定效价之前,先通过方正滴定建立了间接ELISA法的工作条件：抗原最佳包被浓度0.5μg/ml,二抗稀释倍数1：10000。免疫结果显示,不同个体对溶菌酶的敏感不同,兔B2、C1对溶菌酶较为敏感,效价达到了1：150000,相反,兔B1、C2的反应较为迟钝。 在溶菌酶抗原性评估试验中,采用间接ELISA、间接竞争ELISA、Western Blotting等方法检测了兔抗溶菌酶血清与溶菌酶、卵运铁蛋白、卵白蛋白、卵类粘蛋白和α-乳白蛋白之间的免疫反应。试验结果说明,兔抗溶菌酶血清与卵运铁蛋白、卵类粘蛋白之间有非特异性免疫反应,与α-乳白蛋白无任何反应,只与溶菌酶发生特异性反应,具有高度专一选择性。
[Abstract]:Egg lysozyme is a naturally occurring alkaline protease with bactericidal and bactericidal effect, which has been widely used in food industry. However, as a major allergen in eggs, about 35% of egg allergy patients are allergic to lysozyme. In addition, food allergies are a complex network of immune disorders that interact with various antigens and antibodies to identify the interrelationships of various allergens in food allergic reactions. It has important guiding significance for the development of low allergy or non-allergic food. Therefore, in this paper, the high purity egg lysozyme was isolated and purified, and its antigenicity was preliminarily evaluated, which not only provided some technical support for the industrialized production of lysozyme. It can also provide some basic experimental parameters for the development of hypoallergenic or non-allergic egg products. The main contents of this study include the isolation and purification of egg lysozyme, the study of lysozyme activity, the preparation of rabbit anti-lysozyme polyclonal antibody and the evaluation of lysozyme antigenicity. In the process of separation and purification of egg lysozyme, a method of separating lysozyme by CM-Sepharose Fast Flow ion exchange chromatography was established. The results of 5%-15%SDS-PAGE electrophoresis showed that the purity of lysozyme was more than 90% and the recovery rate was about 31. 1%. In the course of lysozyme activity study, some enzyme active substances of egg lysozyme were studied with Bacillus subtilis and its cell wall. The results showed that egg lysozyme could inhibit the growth of Bacillus subtilis obviously. At 50 鈩，

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