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

doi:10.3877/cma.j.issn.1674-1358.2018.02.020

中华实验和临床感染病杂志(电子版) ›› 2018, Vol. 12 ›› Issue (02) : 198 -203. doi: 10.3877/cma.j.issn.1674-1358.2018.02.020

所属专题：文献；

基础论著

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

侯志飞¹, 蒋栋¹, 赵学森¹, 曾辉¹^,^†(

)

^1. 100015　北京，首都医科大学附属北京地坛医院传染病研究所

收稿日期:2017-04-01 出版日期:2018-04-15
通信作者: 曾辉
基金资助:
国家自然科学基金(No. 81571976)

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

Zhifei Hou¹, Dong Jiang¹, Xuesen Zhao¹, Hui Zeng¹^,^†()

^1. Institute of Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing 100015, China

Received:2017-04-01 Published:2018-04-15
Corresponding author: Hui Zeng
About author:
Corresponding author: Zeng Hui, Email: zenghui@ccmu.edu.cn

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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。

关键词: 干扰素诱导跨膜蛋白3, H1N1型流感病毒, 假病毒, 诱导表达, 防御分子

Objective

To 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.

Methods

Full-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).

Results

The 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.

Conclusions

FLP-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.

Key words: Interferon-induced transmembrane protein 3, H1N1 influenza virus, Pseudoviruses, Inducible expression, Restriction factor

图1 HIV/Luc报告基因模式图及荧光素酶活性

图2 IFITM3诱导表达细胞株构建流程图及蛋白表达

图3 IFITM3诱导表达蛋白抑制H1N1型流感假病毒感染

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