摘要
目的构建小鼠无POU域八聚体结合蛋白(non-POU-domain-containing,octamer binding protein,NonO)真核表达载体并在小鼠Hepa 1-6肝癌细胞内表达,观察NonO胞内表达、定位及脂多糖(LPS)对其定位的影响。方法提取小鼠肝组织总RNA,RT-PCR扩增小鼠NonO基因的蛋白编码序列。采用基因重组技术将其亚克隆至pcDNA3-HA真核表达载体中,将该载体瞬时转染Hepa1-6细胞,通过细胞免疫荧光方法观察NonO的胞内表达及LPS对其定位的影响。结果酶切和DNA测序证明所构建质粒正确;细胞免疫荧光可见NonO在Hepa 1-6细胞内表达、分布于细胞核,LPS刺激后NonO分布无明显变化。结论成功构建NonO真核表达载体,为研究NonO在基因表达调控中作用及其生物功能提供了重要载体。进一步证实NonO为定位于细胞核的核蛋白。
Objective To construct the eukaryotic expression vector of mouse non-POU-domain-containing, octamer binding protein (NonO) gene and express it in murine Hepa 1-6 hepatoma cell lines, and to observe the location of NonO and the effect of lipopolysaccharide (LPS) on the location. Methods Total RNA was extracted from mouse liver, and the code sequence of NonO gene was amplified by reverse transcription polymerase chain reaction (RT-PCR). By gene recombination technique, the code sequence of NonO gene was subcloned into eukaryotic expression vector pcDNA3-HA. The vector was transiently transfected into Hepa 1-6 cells, then the expression and subcellular location of NonO was detected by immunofluorescence. Results The eukaryotic expression vector of NonO was correctly constructed, which had been proved by restriction enzyme digestion and DNA sequencing. The plasmids of NonO were expressed and located in nucleus in Hepa 1-6 cells and the location of NonO had not evidently changed after LPS stimulation. Conclusion A eukaryotic expression vector of NonO gene has been successfully constructed. The plasmids will provide an important toll for the further study on the role of NonO in gene expression regulation and biological function. It is further confirmed that NonO is located in nucleus.
出处
《中国现代医药杂志》
2010年第7期1-4,共4页
Modern Medicine Journal of China
基金
国家自然科学基金面上项目(NO.30670828)
