高等学校化学学报2017，Vol.38Issue(5)：770-777,8.DOI:10.7503/cjcu20160794
基于Tet-On 3G的IFITM3诱导表达MDCK细胞系的建立及功能分析
Establishment and Functional Analysis of MDCK Cell Line Induced IFITM3 Expression Based on Tet-On 3G System
摘要
Abstract
To explore the antiviral mechanisms of IFITM3, the eukaryotic expression plasmid pTRE3G- IFITM3 containing IFITM3 gene was constructed based on Tet-On 3G system and then cotranfected with the regulator vector pLVX-Tet3G into MDCK cells.After 48 h, the cells were subjected to select with G418 and puromycin, followed by treatment with or without doxycycline(Dox) to identify IFITM3 expression, continuing to Dox sensitivity analysis, IFITM3+ cell percentage and location analysis.And then, infection by lentiviruses pseudotyped with avian influence virus H5 or H7 hemagglutinin or VSV G proteins was performed to detect luciferase activities.The results indicated that inducible IFITM3-expressing MDCK cell lines were obtained and expression level of IFITM3 was correlated with the dose and induction time of Dox.The concentration of Dox was determined to be 2.5 g/mL, and IFITM3+ cell percentage was up to 75% or more after 12 h.And IFITM3 was distributed in late endosomes/lysosomes and efficiently suppressed the avian influence virus H5 or H7 hemagglutinin or VSV G-mediated viral entry, to lay a foundation for further research into the inhibition mechanism.关键词
干扰素诱导跨膜蛋白3/干扰素刺激基因/诱导表达/病毒进入/抑制Key words
Interferon-inducible transmembrane protein 3/Interferon-stimulated genes/Inducible expression/Virus entry/Restriction分类
化学化工引用本文复制引用
曹婷婷,刘立明,赵翠青,周义发,李昌,金宁一,杜寿文,许汪,邢彬,赵飞,王茂鹏,朱羿龙,白杰英,田宇飞..基于Tet-On 3G的IFITM3诱导表达MDCK细胞系的建立及功能分析[J].高等学校化学学报,2017,38(5):770-777,8.基金项目
国家自然科学基金(批准号:31472197,31402175)、 病原微生物生物安全国家重点实验室开放课题(批准号:SKLPBS1435)、 北京市自然科学基金(批准号:5152023)和吉林省青年科研基金(批准号:20140520173JH)资助.Supported by the National Natural Science Foundation of China(Nos.31472197,31402175),the Open Project of State Key Laboratory of Pathogen and Biosecurity,China(No.SKLPBS1435),the Beijing Natural Science Foundation,China(No.5152023) and the Youth Foundation of Jilin Province,China(No.20140520173JH). （批准号:31472197,31402175）
