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,i TRAQ)结合...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,i TRAQ)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p CMV-MycTMEM43,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p CMV-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。展开更多
The objective of this study was to construct the infectious clone of encephalomyocarditis virus (EMCV) BJC3 strain.The genomic cDNA of the virus was amplified by three overlapping segments using RT-PCR,and cloned into...The objective of this study was to construct the infectious clone of encephalomyocarditis virus (EMCV) BJC3 strain.The genomic cDNA of the virus was amplified by three overlapping segments using RT-PCR,and cloned into low-copy plasmid pWSK29 to construct the full-length cDNA clone pWSKBJC3/ w.The pWSKBJC3/w was in vitro transcribed and transfected into BHK-21 cells to rescue the virus.The results showed that the full-length cDNA clone was infectious and the virus could be rescued in BHK-21 cells.The rescued virus designated RvBJC3W was identified by RT-PCR and indirect immunofluorescence assay(IFA).The rescued virus had similar growth characteristics to its parental virus BJC3 and retained pathogenicity for mice.Our results indicate that the first infectious cDNA clone of EMCV in China has been successfully established and provides an essential tool for investigating the molecular basis of pathogenicity of EMCV.展开更多
文摘【目的】利用网络药理学和分子对接技术发现莪术醇抗脑心肌炎病毒(Encephalomyocarditis virus,EMCV)的作用靶点及机制。【方法】利用PharmMapper、GeneCards数据库获得莪术醇抗EMCV的相关靶点;通过Cytoscape 3.7.2软件、STRING和DAVID数据库构建靶蛋白互作(PPI)网络并筛选关键靶点,对靶点进行GO功能和KEGG通路富集分析,并构建莪术醇抗EMCV靶点-通路网络;通过AutoDock Vina 1.1.2分析莪术醇与靶蛋白的结合能及结合模式。【结果】网络药理学分析结果显示,莪术醇抗EMCV的潜在靶点有9个,其中丝裂原活化激酶14(mitogen-activated protein kinase 14,MAPK14)、信号转导与转录激活因子1(signal transducer and activator of transcription 1,STAT1)、白蛋白(albumin,ALB)、白细胞介素2(interleukin 2,IL2)可能是莪术醇抗EMCV的核心靶点,所得靶点参与C型凝集素受体信号通路、神经营养素信号通路和JAK-STAT信号通路等代谢通路,功能涉及调节炎症反应细胞因子的产生、蛋白激酶活性和药物结合等;分子对接结果显示,4种核心靶蛋白与莪术醇之间存在较强的结合能,均存在疏水形式的结合,其中ALB、STAT1和IL2与莪术醇之间还存在氢键结合。【结论】本研究结果表明,MAPK14、STAT1、ALB和IL2是莪术醇发挥抗EMCV作用的潜在靶点,本研究为莪术醇作为抗EMCV药物的研发提供理论依据和线索。
基金supported by the Key Project of National Natural Science Foundation of China(30530550)
文摘The objective of this study was to construct the infectious clone of encephalomyocarditis virus (EMCV) BJC3 strain.The genomic cDNA of the virus was amplified by three overlapping segments using RT-PCR,and cloned into low-copy plasmid pWSK29 to construct the full-length cDNA clone pWSKBJC3/ w.The pWSKBJC3/w was in vitro transcribed and transfected into BHK-21 cells to rescue the virus.The results showed that the full-length cDNA clone was infectious and the virus could be rescued in BHK-21 cells.The rescued virus designated RvBJC3W was identified by RT-PCR and indirect immunofluorescence assay(IFA).The rescued virus had similar growth characteristics to its parental virus BJC3 and retained pathogenicity for mice.Our results indicate that the first infectious cDNA clone of EMCV in China has been successfully established and provides an essential tool for investigating the molecular basis of pathogenicity of EMCV.
