聚乙二醇修饰对牛乳β-乳球蛋白的致敏性与构象变化的研究

发布时间：2018-01-14 23:26

本文关键词：聚乙二醇修饰对牛乳β-乳球蛋白的致敏性与构象变化的研究　出处：《南昌大学》2012年硕士论文　论文类型：学位论文

【摘要】：聚乙二醇修饰技术(PEGylation)是20世纪七十年代中后期发展起来的一项新技术,通过修饰可以使蛋白药物的抗原性降低或消除、增加水溶性、提高稳定性等一系列优点。本课题研究了PEG修饰对β-乳球蛋白致敏性和构象变化的影响,初步探讨了β-乳球蛋白致敏性与构象变化之间的关系。导致牛乳过敏的主要过敏原之一为β-乳球蛋白(β-lactoglobulin,β-Lg),消除主要致敏原β-乳球蛋白的致敏性始终是国内外乳制品加工技术研究的热点之一。本文以mPEG-SC (20kDa)修饰β-乳球蛋白,通过间接竞争ELISA研究修饰产物抗原性的变化,结果表明：修饰反应时间对β-Lg抗原性降低的效果影响最大,最佳反应条件即修饰反应溶液pH7.0、反应时间8h、mPEG-SC与β-Lg物质的量比3：1时的抗原性为53.64μg/mL (初始值为180μg/mL),抗原性降低率为70.20%; SDS-PAGE得到PEG修饰β-Lg的产物有两种：两点修饰产物(di-PEGylated β-Lg,58kDa)和三点修饰产物(tri-PEGylated β-Lg,78kDa)。通过阳离子交换层析法(树脂为SP Sepharose Fast Flow)分离纯化PEG修饰β-乳球蛋白后的产物,收集洗脱峰。经SDS-PAGE实验得到纯化的产物为两点修饰产物、三点修饰产物。修饰产物的荧光光谱实验结果表明,PEGylation过程中发生荧光淬灭的现象,导致β-Lg的三、四级结构发生了变化,同时β-Lg抗原性的降低伴随着相对荧光强度的降低。圆二色谱分析结果表明,PEGylation过程中,从β-Lg到di-PEGylated β-Lg,再到tri-PEGylatedy β-Lg, β-sheet含量逐渐增加(β-sheet含量:β-Lg33.2%, di-PEGylatedy β-Lg35.7%, tri-PEGylated β-Lg44.6%)；随着结合到β-Lg分子上PEG分子的增加,a-helix从14.3%到14.9%,再到19.2%,表明a-helix略有增加。总之,由构象研究可以说明PEG修饰β-Lg后会使β-Lg构象表位发生变化,导致致敏性发生降低,即PEG修饰β-Lg后可以掩盖β-Lg的抗原决定簇,使蛋白质免疫原性和抗原性产生降低。
[Abstract]:Polyethylene glycol modification ( PEGylation ) is a new technique developed in the middle and late 1970s , and the effect of PEG modification on the sensitization and conformational changes of 尾 - lactoglobulin is studied . The relationship between sensitivity and conformational change of 尾 - lactoglobulin is discussed . One of the main allergens causing milk allergy to milk was 尾 - lactoglobulin ( 尾 - lactoglobulin , 尾 - lg ) . The sensitivity of 尾 - lactoglobulin was modified by indirect competitive ELISA . The results showed that the antigenicity of modified 尾 - lactoglobulin was 53.64 渭g / mL ( initial value was 180 渭g / mL ) . The results showed that the product of PEG - modified 尾 - lg was modified by SDS - PAGE . The results of fluorescence spectrum of modified products show that the three - and four - stage structure of 尾 - lg increased ( 尾 - sheet content : 尾 - Lg33 . 2 % , di - PEGylated 尾 - Lg34.7 % , di - PEGylated 尾 - Lg44.6 % ) . In conclusion , the conformational change can be explained by the conformation study . The conformational epitopes of 尾 - lg can be changed after PEG modification , which results in a decrease in allergenicity , that is , the antigenic determinants of 尾 - lg can be masked after PEG modification of 尾 - lg , which reduces the immunogenicity and antigenicity of the protein .

【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R151

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