本文選題：溶菌酶 + 分離純化��； 參考：《南昌大學》2010年碩士論文

【摘要】： 雞蛋溶菌酶是一種天然存在的具有殺菌抑菌作用的堿性蛋白酶,在食品行業(yè)中得到了廣泛的應用,然而作為雞蛋中的一種主要過敏原,約有35%的雞蛋過敏患者對溶菌酶過敏。另外,食物過敏是一種復雜的網(wǎng)絡免疫失調(diào)癥,多種抗原抗體之間相互作用,探明各種過敏原在食物過敏反應中的相互關(guān)系,對于低過敏或無過敏食品的開發(fā)具有重要的指導意義。因此,本論文分離純化高純度雞蛋溶菌酶,并對其抗原性進行初步評估,這不僅為溶菌酶的工業(yè)化生產(chǎn)提供一定的技術(shù)支持,還能夠為低過敏性或無過敏性蛋制品的開發(fā)提供一些基本的實驗參數(shù)。 本研究的主要內(nèi)容包括雞蛋溶菌酶的分離純化、溶菌酶酶活性質(zhì)研究、兔抗溶菌酶多克隆抗體的制備以及溶菌酶抗原性評估。 在雞蛋溶菌酶分離純化過程中,建立了CM-Sepharose Fast Flow離子交換層析分離溶菌酶的方法。分離得到的樣品經(jīng)5%-15%SDS-PAGE電泳進行鑒定,結(jié)果表明,溶菌酶純度在90%以上,回收率在31.1%左右。 在溶菌酶酶活性質(zhì)研究過程中,以枯草芽孢桿菌及其細胞壁作為研究對象,研究了雞蛋溶菌酶的部分酶活性質(zhì)。研究結(jié)果表明,雞蛋溶菌酶對枯草芽孢桿菌的生長具有明顯的抑制作用,在50℃環(huán)境中,溶菌酶對枯草芽孢桿菌細胞壁具有明顯的降解作用,但是在25℃時的作用并不明顯,這說明溫度對溶菌酶酶活有很大影響。 在兔抗溶菌酶多克隆抗體制備過程中,通過弗氏佐劑來加強免疫,采用皮下多點免疫的方法免疫日本大白兔,分離血清,然后用間接ELISA檢測了兔抗溶菌酶血清效價的變化。在測定效價之前,先通過方正滴定建立了間接ELISA法的工作條件：抗原最佳包被濃度0.5μg/ml,二抗稀釋倍數(shù)1：10000。免疫結(jié)果顯示,不同個體對溶菌酶的敏感不同,兔B2、C1對溶菌酶較為敏感,效價達到了1：150000,相反,兔B1、C2的反應較為遲鈍。 在溶菌酶抗原性評估試驗中,采用間接ELISA、間接競爭ELISA、Western Blotting等方法檢測了兔抗溶菌酶血清與溶菌酶、卵運鐵蛋白、卵白蛋白、卵類粘蛋白和α-乳白蛋白之間的免疫反應。試驗結(jié)果說明,兔抗溶菌酶血清與卵運鐵蛋白、卵類粘蛋白之間有非特異性免疫反應,與α-乳白蛋白無任何反應,只與溶菌酶發(fā)生特異性反應,具有高度專一選擇性。
[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，

本文編號：1908060