β-淀粉样蛋白单体及寡聚体鉴定方法的改进与优化

发布时间：2018-03-02 15:25

本文选题：β-淀粉样蛋白　切入点：电泳　出处：《中国生物化学与分子生物学报》2017年08期 　论文类型：期刊论文

【摘要】：β-淀粉样蛋白(β-amyloid peptide,Aβ)经过自身聚集形成寡聚体,是导致阿尔茨海默病(Alzheimer’s disease,AD)发病的重要风险因素之一。鉴定Aβ寡聚状态对于细胞毒性和寡聚抑制的研究非常重要。本文通过优化Aβ42蛋白用量、增加交联剂、尺寸排阻色谱法(size exclusion chromatography,SEC)和硫磺素T(thioflavin T,Th T)荧光分析等方法,对Aβ42的蛋白质聚集状态进行检测。得到改进和优化的方案:(1)对电泳和电镜的优化,16.5%的Tris-tricine胶,最适合分析Aβ42寡聚状态。使用电泳和电镜对Aβ42检测时,其用量有较严格的限制。电泳的上样量应为0.5μg最佳。50μg/m L Aβ42单体浓度,最适合电镜观察;(2)对Aβ42交联的优化,终浓度为0.3mmol/L BS3,有利于对Aβ42寡聚体的区分;(3)SEC可以利用凝胶孔隙的大小,实现Aβ42单体和寡聚体在非变性条件下的分离。与Aβ40相比,Aβ42进行SEC分析时,单次上样量需达到6.75μg以上;(4)细胞毒性实验和Th T检测,可以反馈Aβ42寡聚状态的鉴定效果。上述检测方法的细化和改进,将为Aβ42肽在AD的研究中提供一定的参考。一些Aβ42特异性寡聚体的鉴定方法本文未提及,仍需要在今后的研究中进一步改进与优化。
[Abstract]:Beta amyloid (-amyloid beta peptide and A beta) after their aggregation form oligomers, is the cause of Alzheimer's disease (Alzheimer 's disease, AD) is one of the important risk factors. Identification of A beta oligomers for cytotoxicity and oligo inhibition is very important. In this paper, through the optimization of A protein beta 42 increase the dosage, crosslinking agent, size exclusion chromatography (size exclusion chromatography SEC T (Thioflavin) and Thioflavin T, Th T) fluorescence analysis method to detect A protein beta 42 aggregation. Improved and optimized the scheme: (1) the optimization of electrophoresis and electron microscope, 16.5% Tris-tricine glue, most suitable for the analysis of A beta 42 oligomeric state of A beta 42. Detection using electrophoresis and electron microscope, the amount is limited. The electrophoresis sample volume should be 0.5 ~ g g/m L best.50 A beta 42 monomer concentration, the most suitable for electron microscopy; (2) the optimization of A beta 42 cross-linked, The final concentration of 0.3mmol/L BS3, is conducive to the distinction of A beta 42 oligomers; (3) SEC can use the gel pore size, separation of A beta 42 monomer and dimer under non denaturing conditions. Compared with A beta 40, beta 42 A SEC analysis, single sample the amount required to reach more than 6.75 mu g; (4) cytotoxicity and Th T detection, the identification results can feedback the A beta 42 oligomeric state. The detection method of refinement and improvement, will be A beta 42 peptide in the AD study to provide some reference. Some methods for the identification of A beta 42 specific oligonucleotide this paper did not mention the dimer, still needs further improvement in future research in optimization.

【作者单位】：西藏民族大学基础医学院形态学教研室;
【基金】：国家自然科学基金项目(No.31660243) 西藏自治区自然科学基金项目(No.12KJZRZMY02)~~
【分类号】：R749.16

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