高等学校化学学报 ›› 2017, Vol. 38 ›› Issue (5): 770.doi: 10.7503/cjcu20160794

• 有机化学 • 上一篇    下一篇

基于Tet-On 3G的IFITM3诱导表达MDCK细胞系的建立及功能分析

曹婷婷1,2, 杜寿文1, 许汪1, 邢彬1, 赵飞1, 王茂鹏1, 朱羿龙1, 白杰英1, 田宇飞1, 刘立明3, 赵翠青3, 周义发2, 李昌1,3,4(), 金宁一1,3,4()   

  1. 1. 军事医学科学院军事兽医研究所, 省部共建吉林省人兽共患病预防与控制重点实验室, 长春 130122
    2. 东北师范大学生命科学学院, 长春 130022
    3. 温州大学病毒学研究所, 温州 325035
    4. 江苏省动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009
  • 收稿日期:2016-11-16 出版日期:2017-05-10 发布日期:2017-04-20
  • 作者简介:联系人简介: 金宁一, 男, 博士, 研究员, 博士生导师, 中国工程院院士, 主要从事分子病毒学与免疫学研究. E-mail:ningyik@126.com;李 昌, 男, 博士, 副研究员, 主要从事病毒感染与免疫研究. E-mail:lichang78@163.com
  • 基金资助:
    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

Establishment and Functional Analysis of MDCK Cell Line Induced IFITM3 Expression Based on Tet-On 3G System

CAO Tingting1,2, DU Shouwen1, XU Wang1, XING Bin1, ZHAO Fei1, WANG Maopeng1, ZHU Yilong1, BAI Jieying1, TIAN Yufei1, LIU Liming3, ZHAO Cuiqing3, ZHOU Yifa2, LI Chang1,3,4,*(), JIN Ningyi1,3,4,*()   

  1. 1. Military Veterinary Institute, Academy of Military Medical Sciences, Key Labtoratory of Jinlin Province for Zoonosis Prevention and Control, Changchun 130122, China
    2. College of Life Sciences, Northeast Normal University, Changchun 130022, China
    3. Institute of Virology, Wenzhou University, Wenzhou 325035, China
    4. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou 225009, China
  • Received:2016-11-16 Online:2017-05-10 Published:2017-04-20
  • Contact: LI Chang,JIN Ningyi E-mail:lichang78@163.com;ningyik@126.com
  • Supported by:
    † 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)

摘要:

利用Tet-On 3G系统构建了含人IFITM3基因的真核表达质粒pTRE3G-IFITM3, 并将其与调控质粒pLVX-Tet3G共转染犬肾细胞(MDCK), 转染后48 h用G418和嘌呤霉素进行筛选, 用多西环素(Dox)对获得的细胞系进行诱导表达鉴定, 并进行Dox敏感性分析、 IFITM3+细胞百分数及定位分析. 用含萤光素酶(Luciferase)报告基因的禽流感病毒H5/H7蛋白或VSV G蛋白包裹的假型慢病毒颗粒进行感染实验, 检测萤光素酶活性. 结果表明, 筛选获得了携带人IFITM3的MDCK诱导表达细胞系, 且IFITM3表达量与Dox剂量和诱导时间相关; 确定Dox工作浓度为2.5 μg/mL, 诱导12 h时IFITM3+细胞数达75%以上, 且IFITM3在晚期内体/溶酶体存在分布; 假型病毒感染及萤光素酶活性分析表明, IFITM3可显著抑制禽流感病毒H5, H7和VSV G蛋白介导的病毒进入, 为深入探究其具体的抑制机制奠定了基础.

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

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.

Key words: Interferon-inducible transmembrane protein 3, Interferon-stimulated genes, Inducible expression, Virus entry, Restriction

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