诱导表达人IFITM3的293细胞株的建立及其对H1N1型流感病毒侵染作用

目的建立诱导表达IFITM3蛋白的293细胞株，为进一步研究人IFITM3对H1N1型流感病毒侵染的作用及其分子机制提供细胞模型。 方法构建IFITM3/pcDNA5/FRT/TO expression vector质粒及诱导表达细胞株，建立HN1型流感病毒假病毒评价系统，利用蛋白印迹（Western blot）和荧光素酶报告基因对筛选的诱导表达细胞株功能进行检测。 结果建立的293诱导表达细胞株能够很好表达目的蛋白，且诱导表达出来的IFITM3蛋白使H1N1型流感假病毒感染率下降97%（t = 38.08、P < 0.001），使水泡性口炎假病毒（VSVpp）感染率下降68%（t = 54.56、P < 0.001），而阴性对照组小鼠白血病假病毒（MLVpp）感染率（t = 1.282、P = 0.2208）和拉沙假病毒（LASVpp）感染率（t = 0.4814、P = 0.6377）无显著影响。 结论IFITM3蛋白诱导表达细胞株可以显著抑制H1N1型流感病毒感染，为进一步深入研究IFITM3抑制流感病毒感染的作用机制和分子机理提供了细胞模型和实验基础。

Establishment of 293 cell line inducibly expressing human IFITM3 and its inhibition to viral entry of H1N1 influenza virus

ObjectiveTo establish FLP-IN T Rex 293 cell line inducibly expressing human interferon-inducible transmembrane protein 3 (IFITM3) and provide a cell model to explore the mechanism of IFITM3 inhibiting vial entry of influenza virus. MethodsFull-length IFITM3 cDNA was cloned into pcDNA5/FRT/TO expression vector. Then 293 cell line inducibly expressing human IFITM3 was established by co-transfection of IFITM3/pcDNA5/FRT/TO expression vector and pOG44 plasmid and following antibiotic screening. The inducible expression of IFITM3 in 293 cells was tested by Western blot and immunofluorescence. The inhibitory effect of viral entry by IFITM3 was examined with H1N1 influenza virus pesudotyped virus (H1N1pp). ResultsThe established FLP-IN T Rex 293 cell line of IFITM3 was able to express the target protein well, and the induced IFITM3 protein decreased the infection rate of H1N1 influenza pseudovirus by 97% (t = 38.08, P < 0.001), and the infection rate of vesicular stomatitis pseudovirus (VSVpp) decreased by 68% (t = 54.56, P < 0.001). Negative control group mice leukemia pseudovirus (MLVpp) infection rate (t = 1.282, P = 0.2208) and Lashapseudovirus (LASVpp) infection rate (t = 0.4814, P = 0.6377) had no significant influence. ConclusionsFLP-IN T Rex 293 cell line inducibly expressing human IFITM3 is a pivotal restriction factor inhibiting H1N1 influenza virus entry. This IFITM3-inducible expression cell line can provide a useful platform to study IFITM3 antiviral mechanism.