目的 探讨跨膜蛋白43(transmembrane protein 43,TMEM43)在脑心肌炎病毒(encephalomyocarditis virus,EMCV)感染过程中的作用.方法 采用同位素标记相对和绝对定量(isobaric tags for relative and absolute quantitation,iTRAQ)结合液相色谱-串联质谱(liquid chromatography-tandem mass spectrometry,LC-MS/MS)高通量蛋白质组学技术,对EMCV感染的BHK-21细胞进行蛋白质组学分析;采用分子克隆技术构建人源TMEM43重组质粒pCMV-Myc-TMEM43,转染HEK293细胞后,感染EMCV(MOI=3),real-time PCR法检测TMEM43过表达对EMCV体外增殖以及EMCV吸附和进入的影响;RNAi试验筛选最有效干扰序列,转染HEK293细胞后,感染EMCV(MOI=3),real-time PCR法检测TMEM43下调对EMCV体外增殖的影响.结果 EMCV感染的BHK-21细胞TMEM43表达显著增加.重组质粒pCMV-Myc-TMEM43可在HEK293细胞正常表达,TMEM43过表达对EMCV复制有显著促进作用,病毒感染滴度也有一定提高;TMEM43过表达在病毒进入阶段发挥促进作用,而对吸附过程无任何影响.TMEM43下调对EMCV增殖有明显抑制作用.结论 TMEM43促进EMCV复制增殖,主要在病毒进入阶段发挥作用.
Effect of transmembrane protein 43 on proliferation of encephalomyocarditis virus in vitro
Objective To investigate the role of transmembrane protein 43(TMEM43)in the process of encephalomyoca-rditis virus(EMCV)infection.Methods The high-throughput proteomics using isobaric tags for relative and absolute quan-titation(iTRAQ)coupled with liquid chromatography-tandem mass spectrometry(LC-MS/MS)was used to analyze the samples prepared from EMCV infected BHK-21 cells.The human TMEM43 gene recombinant plasmid pCMV-Myc-TMEM43 was constructed by molecular cloning technique and transfected into HEK293 cells,which were subjected to EMCV(MOI=3),and detected for the effect of TMEM43 overexpression on EMCV proliferation and viral adsorption and entry in vitro by real-time PCR.RNAi was performed to screen the most efficient sequence targeting TMEM43,which was transfected into HEK293 cells.After infection with EMCV(MOI=3),the cells were detected for the effect of TMEM43 knockdown on EMCV proliferation and viral adsorption and entry in vitro by real-time PCR.Results TMEM43 expression in BHK-21 cells infected with EMCV significantly increased.The recombinant plasmid pCMV-Myc-TMEM43 was normally expressed in HEK293 cells,and the overexpression of TMEM43 significantly promoted the replication of EMCV and increased the virus titer to some extent.Further studies showed that the overexpression of TMEM43 played an important role in the entry stage of the virus,but had no effect on the adsorption process.Down-regulation of TMEM43 expression significantly inhibited the proliferation of EMCV.Conclusion TMEM43 promotes the replication and proliferation of EMCV,and mainly plays an im-portant role in the virus entry process.
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