人高迁移率族蛋白B1细胞内定位及移位研究

Study of localization and translocation of human high mobility group protein B1 in eukaryotic cells

目的观察人高迁移率族蛋白B1(HMGB1)在真核细胞内的定位和移位情况。方法构建人HMGB1及增强型绿色荧光蛋白(EGFP)融合蛋白的哺乳动物细胞表达载体。通过两步亚克隆的方法,将HMGB1和EGFP的编码序列以融合表达形式克隆到带有血凝素(hytohemagglutinin,HA)标记的载体pcDNA3HA上,随后转染293A细胞,在荧光显微镜下观察结果。结果重组质粒经酶切、聚合酶链反应(PCR)和测序鉴定证明构建正确,并在293A细胞中得到大量表达。荧光显微镜观察发现,融合蛋白主要分布于细胞核中,经肿瘤坏死因子α(TNFα)刺激后18h,部分细胞中融合蛋白从胞核移位到胞质,与预期结果一致。结论HMGB1的绿色荧光蛋白融合表达载体构建成功。该载体能在哺乳动物细胞中有效表达并正确定位、移位,为下一步深入研究HMGB1作用细胞的信号通路提供了一个重要的工具。

Objective To observe the localization and transloeation of human high mobility group protein B1 （HMGB1） protein in eukaryotic cells. Methods The vector that expressed the fusion protein of human HMGB1 and enhanced green fluorescent protein （EGFP） in mammalian cells were constructed. HMGB1 and EGFP were subcloned into hytohemagglutinin （HA） tagged vector pcDNA3- HA by two steps, the construct of which was then transfected into 293A cells and observed with fluorescence microscope. Results The recombinant plasmid was verified by enzyme digestion, polymerase chain reaction （PCR） and sequence analysis, and the fusion protein was highly expressed in 293A cells. With fluorescence microscopy the green fluorescence was found to be distributed in the nuclei. Eighteen hours after the stimulation with tumor necrosis factor - α （TNF - α）, it was found to have translocated into the cytoplasm in some of the cells. Conclusion The expression vector for HMGB1-EGFP fusion protein is successfully constructed and effectively expressed in mammalian cells, and it can serve as an important tool in the study of the signal pathway of HMGB1 in mammalian cells.