应用G3BP1稳转细胞系监测应激状态下的应激颗粒形成

doi:10.11843/j.issn.0366-6964.2020.09.025

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (9): 2275-2283.doi: 10.11843/j.issn.0366-6964.2020.09.025

应用G3BP1稳转细胞系监测应激状态下的应激颗粒形成

邵琪¹, 屈阳², 朱子晨³, 孟春春¹, 仇旭升¹, 廖瑛¹, 谭磊¹, 宋翠萍¹, 刘炜玮¹, 孙英杰^1*, 丁铲^1*

1. 中国农业科学院上海兽医研究所, 上海 200241;
2. 西北农林科技大学动物医学院, 杨凌 712100;
3. 山东农业大学动物科技学院, 泰安 271018

收稿日期:2020-03-02 出版日期:2020-09-25 发布日期:2020-09-25
通讯作者: 丁铲,主要从事家禽病毒性传染病基础研究与防控技术研究,E-mail:shoveldeen@shvri.ac.cn;孙英杰,主要从事抗病毒先天性免疫及应激反应研究,E-mail:sunyingjie@shvri.ac.cn
作者简介:邵琪(1996-),女,江西九江人,硕士生,主要从事动物病毒分子生物学研究,E-mail:2052394856@qq.com
基金资助:
国家自然科学基金面上项目（31872453）；国家重点研发计划项目（2017YFD0500800；2018YFD0500100）

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

SHAO Qi¹, QU Yang², ZHU Zichen³, MENG Chunchun¹, QIU Xusheng¹, LIAO Ying¹, TAN Lei¹, SONG Cuiping¹, LIU Weiwei¹, SUN Yingjie^1*, DING Chan^1*

1. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China;
2. College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China;
3. College of Animal Science and Veterinary Medicine, Shandong Agricultural University, Tai'an 271018, China

Received:2020-03-02 Online:2020-09-25 Published:2020-09-25

摘要/Abstract

摘要： 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。

关键词: G3BP1, 应激颗粒, 新城疫病毒, 病毒感染

Abstract: 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.

Key words: G3BP1, stress granule, Newcastle disease virus, virus infection

中图分类号:

S855.3

邵琪, 屈阳, 朱子晨, 孟春春, 仇旭升, 廖瑛, 谭磊, 宋翠萍, 刘炜玮, 孙英杰, 丁铲. 应用G3BP1稳转细胞系监测应激状态下的应激颗粒形成[J]. 畜牧兽医学报, 2020, 51(9): 2275-2283.

SHAO Qi, QU Yang, ZHU Zichen, MENG Chunchun, QIU Xusheng, LIAO Ying, TAN Lei, SONG Cuiping, LIU Weiwei, SUN Yingjie, DING Chan. Monitoring of Stress Granule Formation under Stress by G3BP1 Stable Expressing Cell Line[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(9): 2275-2283.

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应用G3BP1稳转细胞系监测应激状态下的应激颗粒形成

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

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