CAJ | 학술논문

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。
목적：탐토 HaloTag 기술용우α-돌촉핵단백질(α-synuclein,SNCA)재세포내형광성상급반정량분석적방법급기응용。방법채용중조극륭기술구건 pHalo-SNCA 질립,장기전염지 HEK293인배신세포중,경 Western blotting 감정 Halo-SNCA 융합단백질적표체；통과격광공취초현미경관찰 Halo-SNCA 재세포내적정위여분포；통과단백매최화적해우련반응적형광강도측정세포내 Halo-SNCA 적형광강도진행목적단백질적간접정량분석。결과본연구성공구건료 Halo-SNCA 진핵표체질립,능구재전염적진핵세포중고효표체목적융합단백질,해융합단백질결합특정적형광배기후,가통과격광공취초현미성상분석 Halo-SNCA 재세포중적동태수평여분포상황。차외,경수해석방적형광배기가용우표체융합단백질재세포중교위정학적상대정량분석。결론HaloTag 기술능구용우 SNCA 재세포내성상화정량분석,동시가이작위세포생물학화생물화학분석적가관련검측지표。대우SNCA 적생리공능이급재신경계통병변중적작용등상관연구,HaloTag 구유령인촉목적잠재응용전경。
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.