干扰素膜蛋白Tet-on系统稳定细胞系的建立及对CA16病毒的抑制作用

Establishment of Controllable Interferon Membrane Protein-expressing HeLa Cell Line that Restricts CA16 Virus Replication

通过Tet-on调控系统,构建受多西环素诱导表达干扰素诱导的跨膜蛋白(interferon-induced transmembrane proteins 1/2/3,IFITM1/2/3)基因的HeLa细胞系,并初步探索了IFITM蛋白对柯萨奇病毒A16(CA16)的抑制作用.首先将调控质粒pTet-on转染进入HeLa细胞,通过G418筛选出阳性克隆细胞系,在此细胞系基础上共同转染反应质粒pTRE2-IFITM1/2/3和伴侣质粒pTK-Hyg,通过潮霉素筛选出单克隆细胞系,加入多西环素后利用Western印迹筛选出可诱导表达IFITM1/2/3蛋白的单克隆细胞系.使用实时荧光定量PCR(RT-qPCR)检测发现,多西环素诱导表达的IFITM蛋白对不同感染复数(multiplicity of infection,MOI)的CA16具有明显的抑制作用,其中IFITM 3对CA16的抑制效果最为明显.Tet调控IFITM1/2/3基因表达HeLa细胞系的成功建立,为进一步研究IFITM基因的功能及其抗病毒机理提供了一个理想的细胞模型.

This study is to establish HeLa cell line controllably expressing IFITM1/2/3 genes utilizing Tet-on system and explore the antiviral effect of IFN-induced transmembrane （IFITM） proteins on replication of coxsackievirus A16（CA16 virus）. Plasmid pTet-on was transfected and sifted by G418 to generate HeLa-tet cell line. Each pTRE2-IFITM plasmid companied with pTK-Hyg plasmid was cotransfected into positive clones followed by hygromycin B selection to generate HeLa/Tet/pTRE2- IFITM1/2/3 cells. The positive cell line was verified by Western blotting analysis to detect the expression of IFITM1/2/3 proteins after doxycycline treatment. Controllable IFITM-expressing HeLa cell line was infected with different multiplicity of infection （MOI） of CA16 virus, then the level of virus RNA was detected by real-time quantitative PCR （RT-qPCR） to determine the inhibitory effect of each IFITM protein. The results demonstrated that IFITM proteins displayed strong restriction effect on CA16 virus replication especially IFITM3. In this study we established the IFITM1/2/3 controllable expressing HeLa cell line and provided suitable cell model for further study in the function and antiviral mechanism of IFITM proteins.