TRIM22稳定过表达细胞系的构建及其抗流感病毒功能

基础医学与临床 ›› 2016, Vol. 36 ›› Issue (5): 682-687.

TRIM22稳定过表达细胞系的构建及其抗流感病毒功能

陈志高¹,李春峰²,秦晓峰³,程根宏³

1. 中国医学科学院
2. 中国医学科学院基础医学研究所
3. 中国医学科学院基础医学研究所北京协和医学院基础学院

收稿日期:2016-01-19 修回日期:2016-03-16 出版日期:2016-05-05 发布日期:2016-04-26
通讯作者: 程根宏 E-mail:gcheng@mednet.ucla.edu

Construction of stably expressing TRIM22 HEK293T cell line and its role of inhibiting influenza virus replication

Received:2016-01-19 Revised:2016-03-16 Online:2016-05-05 Published:2016-04-26

摘要/Abstract

摘要： 目的构建稳定表达TRIM22的HEK293T细胞系HEK293T-TRIM22，观察TRIM22蛋白在该细胞内的表达及对流感病毒复制的影响并初步探究其抑制机制。方法扩增TRIM22基因并与反转录病毒载体进行体外重组连接，经菌液PCR、测序确认后与反转录病毒骨架载体共转染进HEK293T细胞进行包装病毒。用包装的反转录病毒感染HEK293T细胞，感染24h后用嘌呤霉素筛选稳定过表达TRIM22的细胞，筛选48 h后用Western blot检测TRIM22表达水平；用IAV-LUC病毒检测稳定过表达TRIM22的HEK293T对流感病毒的抑制作用；再用双分子荧光互补实验（BiLC）检测与TRIM22相互作用的流感蛋白。结果经测序确认目的基因TRIM22成功克隆到载体pQC-XIP中。包装反转录病毒并感染HEK293T细胞，经过嘌呤霉素筛选后，稳定过表达TRIM22的HEK293T细胞中TRIM22的蛋白表达水平升高(P<0.05)。在稳定过表达TRIM22的细胞内流感病毒复制减弱(P<0.05)。BiLC检测TRIM22与流感蛋白NP有相互作用。结论稳定过表达TRIM22的HEK293T细胞系构建成功，TRIM22与NP有相互作用并且抑制流感病毒复制。

关键词: TRIM22, 逆转录病毒, HEK293T细胞, 流感病毒

Abstract: Objective To construct stably expressing TRIM22 HEK293T cell line and investigate the inhibitory function of TRIM22 to influenza A virus replication. Screen the IAV proteins which can interact with TRIM22. Methods Human TRIM22 gene was cloned onto pQC-XIP with FLAG tag using PCR. Then, the recombinant plasmid and pVSV-G, pGag-Pol were co-transfected into HEK293T cells for packaging retrovirus. Packaged retrovirus was collected and used to infect HEK293T cell, followed by screening with Puromycin. Finally, expression of TRIM22 was detected by Western blot. The inhibitory function of TRIM22 to influenza virus replication was detected with IAV-LUC. The interactions between IAV proteins and TRIM22 were detected by BiLC. Results Gene sequencing indicated that pQC-XIP-TRIM22 was successfully constructed. TRIM22 protein was detected in lysates of stable cell line. Contrast to stable cell line expressing EGFP, that expressing TRIM22 could inhibit the Influenza virus replication. TRIM22 can bind to NP. Conclusions Stable cell line expressing TRIM22 was successfully constructed. TRIM22 can inhibit the influenza virus and it can interact with NP.

Key words: TRIM22, retrovirus, HEK293T, influenza virus

中图分类号:

R392-33

陈志高李春峰秦晓峰程根宏. TRIM22稳定过表达细胞系的构建及其抗流感病毒功能[J]. 基础医学与临床, 2016, 36(5): 682-687.