带标签的高迁移率蛋白N2(HMGN2)重组质粒用于亚细胞定位分析

Application of HMGN2-tag Constructs to Analysis of HMGN2 Distribution in HeLa Cells

为进行HMGN2的亚细胞定位分析,提取人LAK细胞总RNA,设计合成相应引物,应用RT-PCR从其总RNA中扩增HMGN2 cDNA,以pcDNA3.1-myc-his和pEGFP-N1为载体,成功构建出HMGN2真核表达载体,转染HeLa细胞,转染效率达70%～80%,用抗六组氨酸臂的单克隆抗体经免疫细胞化学染色显示经pcDNA3.1-myc-his-HMGN2转染的HeLa细胞除细胞核外,胞浆亦明显着色,未转染的Hela细胞无论细胞核还是细胞浆均显阴性;用兔抗人HMGN2多克隆抗体进行的免疫细胞化学染色显示转染的HeLa细胞与用抗六组氨酸臂的单克隆抗体检测结果相同,而未转染的Hela细胞核内和胞浆也均有着色.用两种抗体行ELISA分析在细胞培养上清亦检测到HMGN2.激光共聚焦显微镜下观察pEGFP-N1-HMGN2转染的HeLa细胞发现绿色荧光主要在核内,但核外也同样存在.本实验结果证明HMGN2不仅存在于细胞核中,而且分布于细胞浆,亦可分泌到细胞外.

This study sought to clarify the distribution of HMGN2 in HeLa cells. The recombinant eukaryotic expression vectors pcDNA3. 1-myc-his-HMGN2 and pEGFP-N1-HMGN2 were constructed, and then were transfected into HeLa cells, immunocytochemistry staining indicated that HMGN2 were present not only in HeLa nucleus but also in the cytoplasm. The presence of HMGN2 was also detected in the culture supernatant by ELISA with rabbit anti-serum against HMGN2 and mouse anti-His6 monoclonal antibodies. The confocal microscope observation showed the same subcellular localization as that of immunocytochemistry staining. There results suggested that HMGN2 could be present in the nucleus and cytoplasm of HeLa cell as well as in the extracellular environment.