摘要
目的观察鼠高迁移率族蛋白B2(HMGB2)在真核细胞中的定位和移位情况。方法提取BALB/c小鼠肝脏组织总RNA,通过RT-PCR扩增得到HMGB2编码序列。然后将该编码序列克隆到带有血凝素(HA)标记的载体pcDNA3-HA上,构建质粒命名为pcDNA3-HA-HMGB2,随后将其转染NIH3T3细胞。荧光显微镜观察仅转染质粒的细胞和质粒转染与亚砷酸钠刺激30min和1h后细胞内HA-HMGB2融合蛋白的定位及移位情况。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NIH3T3细胞中得到大量表达。荧光显微镜观察发现,HA-HMGB2蛋白主要分布于细胞核中,经亚砷酸钠刺激后30min,部分细胞中的蛋白从胞核移位到胞质,刺激1h后蛋白则主要分布于胞质中。结论成功构建带HA标签的HMGB2真核表达载体。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HMGB2作用细胞的信号通路提供了一个重要工具。
Objective To observe the localization and transposition of mouse high mobility group protein B2 （HMGB2） in eukaryotic cells. Methods The total RNA was extracted from livers of BALB/c mice, the corresponding coding sequences for mouse HMGB2 （Gen Bank accession No 008252） were amplified by RT-PCR and cloned into hemaggiutinin （HA）-tagged vector pcDNA3-HA. The construct was then transfected into NIH 3T3 cells, and the localization and transposition of mouse high mobility group protein B2 were observed with fluorescence microscope. The recombinant plasmid was verified by enzyme digestion, polymerase chain reaction （PCR） and sequence analysis. Results The fusion protein was highly expressed in NIH 3T3 cells. With fluorescence microscopy the fusion protein was found to be distributed in the nuclei. Thirty minutes after the stimulation with arsenite, the fusion protein was found to have been transposited into the cytoplasm in some of the cells, and one hour after stimulation, it was found to be located in cytoplasm. Conclusion The expression vector for HA HMGB2 fusion protein is successfully constructed and effectively expressed in mammalian cells, and it can serve as an important tool in the study on the signal pathway of HMGB2 in mammalian cells.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2009年第4期434-436,444,共4页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金资助项目(30572151
30670828)
国家自然科学基金委-广东省人民政府自然科学联名基金重点项目(U0632004)
教育部长江学者和创新团队发展计划项目(IRT0731)
