应用G3BP1稳转细胞系监测应激状态下的应激颗粒形成

哺乳动物细胞受到应激刺激后，会在细胞中形成致密的颗粒状结构，该结构被称为应激颗粒（stress granules，SG），其中G3BP1是应激颗粒重要的组成成分和标志蛋白。为了动态监测SG的形成情况，构建稳定表达GFP-G3BP1蛋白的HeLa细胞系。首先，扩增G3BP1基因，并将其克隆到慢病毒载体中，从而获得重组质粒Lenti-GFP-G3BP1，利用三质粒慢病毒包装系统包装为表达GFP-G3BP1蛋白的慢病毒颗粒，并感染HeLa细胞，通过嘌呤霉素初步筛选阳性细胞，随后，采用有限稀释法筛选出稳定表达GFP-G3BP1蛋白的HeLa单克隆细胞系，并采用Western blot和直接免疫荧光方法检测细胞系中GFP-G3BP1蛋白的表达及功能。结果发现，在荧光显微镜下可以观察到明显的G3BP1细胞质特异性荧光，Western blot结果显示GFP-G3BP1特异性条带，说明稳定表达GFP-G3BP1的HeLa细胞系构建成功。最后，作者利用亚砷酸钠/热休克/新城疫病毒处理细胞系后对GFP-G3BP1表达形态进行动态监测，证实了细胞受到刺激后，SG逐渐积累的过程。该细胞和相关动态监测方法为后续SG和新城疫病毒的相关研究奠定了基础。

Monitoring of Stress Granule Formation under Stress by G3BP1 Stable Expressing Cell Line

When mammalian cells are stimulated by stress, multiple dense granular structures form in cells. These structures are called stress granules (SGs). G3BP1 is an important component and marker protein of stress granules. To dynamically monitor the formation of SG, the HeLa cell line stably expressing GFP-G3BP1 was constructed. The G3BP1 gene was amplified and cloned into a lentiviral vector to obtain a recombinant plasmid Lenti-GFP-G3BP1. The three-plasmid lentiviral packaging system was used to package the lentiviral particles expressing GFP-G3BP1, which were used to infect HeLa cells. The positive cells were initially selected by puromycin, and further selected by limited dilution to get the monoclonal cells stably expressing GFP-G3BP1. The function and expression of GFP-G3BP1 in the cell line were detected by Western blot and immunofluorescence assays. The obvious cytoplasm-specific fluorescence of G3BP1 was observed. Western blot results show a GFP-G3BP1 specific band, indicating that the HeLa cell line stably expressing GFP-G3BP1 was constructed successfully. Finally, the formation of SG in response to ARS/heatshock/Newcastle disease virus treatment were dynamically monitored. The results showed the gradual accumulation of SGs in response to these treatments. The specific cell line and method we established have laid the foundation for subsequent research on SG and Newcastle disease virus.