摘要
目的探讨HaloTag技术用于α-突触核蛋白质(α-synuclein,SNCA)在细胞内荧光成像及半定量分析的方法及其应用。方法采用重组克隆技术构建pHalo-SNCA质粒,将其转染至HEK293人胚肾细胞中,经Western blotting鉴定Halo-SNCA融合蛋白质的表达;通过激光共聚焦显微镜观察Halo-SNCA在细胞内的定位与分布;通过蛋白酶催化的解偶联反应的荧光强度测定细胞内Halo-SNCA的荧光强度进行目的蛋白质的间接定量分析。结果本研究成功构建了Halo-SNCA真核表达质粒,能够在转染的真核细胞中高效表达目的融合蛋白质,该融合蛋白质结合特定的荧光配基后,可通过激光共聚焦显微成像分析Halo-SNCA在细胞中的动态水平与分布状况。此外,经水解释放的荧光配基可用于表达融合蛋白质在细胞中较为精确的相对定量分析。结论HaloTag技术能够用于SNCA在细胞内成像和定量分析,同时可以作为细胞生物学和生物化学分析的可关联检测指标。对于SNCA的生理功能以及在神经系统病变中的作用等相关研究,HaloTag具有令人瞩目的潜在应用前景。
Objective To develop a novel method for intracellular imaging and semi-quantification of alpha-synuclein（SNCA） using HaloTag technology by staining of fluorescent fusion proteins. Methods The mammalian expression vector of a Halo-SNCA was constructed, and the obtained plasmid was transfected into HEK293 cells. The expressed fusion protein was identified by Western blotting, and meanwhile labeled with TMR fluorescent ligand for laser confocal microscopy. The samples following imaging were subjected to protease digestion; the released ligands were quantified according to the measurements of fluorescence intensities to evaluate the expression levels of Halo-SNCA. Results The Halo-SNCA fusion protein was efficiently expressed in eukaryotic cells. The intracellular distribution of the Halo-SNCA was demonstrated by confocal microscopy with high resolution. Labeling with HaloTag fluorescent ligand was a practical approach to semi-quantitatively estimate the expression levels of Halo-SNCA with TMR fluorescent intensities. Conclusion The pHalo-SNCA vector can be a useful tool to analyze SNCA intracellular functions by simultaneously monitoring the localization/distribution and the dynamics of expression levels.
出处
《首都医科大学学报》
CAS
2014年第3期324-330,共7页
Journal of Capital Medical University
基金
国家自然科学基金(81372284
81201816)~~
