蛋白质检测与相互作用的毛细管电泳新方法研究

发布时间：2018-04-29 01:17

本文选题：毛细管电泳 + 前沿分析法　；参考：《中国人民解放军军事医学科学院》2010年硕士论文

【摘要】： 高灵敏蛋白质检测方法及相互作用研究对疾病的诊治具有重要意义,同时也是蛋白质组学得以迅速发展的巨大动力。近年来,针对功能蛋白的高灵敏度分析检测新方法研究,一直是生命科学研究中蛋白质分析领域的研究热点。本论文即开展了基于毛细管电泳(CE)技术的相关蛋白质间接检测新方法及与小分子药物或单链DNA适配体的结合作用研究,全文共分为四章。第一章综述了蛋白质的高灵敏度检测与识别技术概况,重点总结了近年来CE技术在蛋白质分离分析、相互作用中的相关进展。第二章中,我们首次将具有高效分离能力的CE方法和BCA法结合起来,建立了一种间接检测蛋白质的新方法。以β-环糊精为包合添加剂,有效地实现了BCA-Cu+复合物和过量游离BCA分子的分离,通过微波辅助反应达到BCA-Cu+快速、完全显色的目的,实现了在低波长处对转铁蛋白、蓖麻毒素等功能蛋白的快速、高灵敏度检测的目的。本方法亦成功应用于第一届蓖麻毒素国际实验室间比对测试(2009年)蓖麻毒素蛋白样品的检测中。所测定的样品中蓖麻毒素的含量结果与真值相比,偏差介于0.40-6.8%之间,说明了我们在CE中以BCA-Cu+复合物浓度的定量间接指代蛋白质定量之思路的正确性。第三章通过CE-前沿分析法(CE-FA)研究碱性药物CBZ与HSA间相互作用,注重选择R值处于较窄范围的不同浓度的CBZ-HSA体系进行测定,从而使线性方程与非线性方程所得到的结合参数符合较好,方便地求出了该体系的结合常数和结合比,该弱相互作用体系的解离常数(Kd)值为7.09×10-5M,CBZ与HSA近似为1:1结合,CBZ结合到蛋白上的结合百分数介于15.8-22.4%之间。第四章使用CE-FA法,结合激光诱导荧光检测和适配体标记技术,以多种方式(R-Cf非线性拟合、平台峰高变化-浓度间的非线性拟合、单点测定法)深入探讨和表征了凝血酶适配体F-29mer和凝血酶间的结合作用,并详细讨论了三种方法所各适用的范围。
[Abstract]:The study of highly sensitive protein detection and interaction is of great significance to the diagnosis and treatment of diseases, and it is also a huge driving force for the rapid development of proteomics. In recent years, high sensitivity analysis and detection of functional proteins has been a hot topic in the field of protein analysis in life sciences. In this paper, a new method for indirect detection of related proteins based on capillary electrophoresis (CE) technique and its binding to small molecular drugs or single-stranded DNA aptamers were studied. The whole thesis is divided into four chapters. In the first chapter, the high sensitivity detection and recognition technology of protein is reviewed, and the progress of CE in protein separation and interaction is emphatically summarized. In the second chapter, we combine CE method with BCA method for the first time, and establish a new method for the indirect detection of protein. Using 尾 -cyclodextrin as inclusion additive, the separation of BCA-Cu complex and excessive free BCA molecules was realized effectively. The aim of rapid and complete color development of BCA-Cu was achieved by microwave-assisted reaction, and transferrin at low wavelength was realized. The aim of rapid and high sensitivity detection of ricin and other functional proteins. This method has also been successfully applied to the determination of ricin samples in the first International Laboratory comparison Test of ricin (2009). Compared with the true value, the deviation of the determination of ricin content in the sample is between 0.40-6.8%, which indicates the correctness of our idea of quantifying protein indirectly with the concentration of BCA-Cu complex in CE. In chapter 3, the interaction between CBZ and HSA was studied by CE-frontier analysis method, and the determination of CBZ-HSA with different concentrations in a narrow range of R values was emphasized. Thus, the binding parameters obtained by linear equation and nonlinear equation are in good agreement, and the binding constant and binding ratio of the system are obtained conveniently. The dissociation constant (Kd) of the weak interaction system is 7.09 脳 10 ~ (-5) M ~ (-1) C _ (Z) and HSA is approximately 1:1 bound to protein. The percentage of binding to protein is between 15.8-22.4%. In chapter 4, we use CE-FA method, combined with laser-induced fluorescence detection and aptamer labeling, to fit the R-Cf nonlinearity in many ways, and the nonlinear fitting between peak height of platform and concentration. The binding between thrombin aptamer F-29mer and thrombin was investigated and characterized, and the suitable ranges of the three methods were discussed in detail.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R341

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