基于Tet-On 3G的IFITM3诱导表达MDCK细胞系的建立及功能分析

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2.5μg/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.

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

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.