干扰素诱导跨膜蛋白2对脑心肌炎病毒体外增殖的影响

EEffect of interferon-induced transmembrane protein 2 on proliferation of encephalomyocarditis virus in vitro

目的探讨干扰素诱导跨膜蛋白2(interferon induced transmembrane proteins 2,IFITM2)对脑心肌炎病毒(encephalomyocarditis virus,EMCV)体外增殖的影响。方法将重组表达质粒pcDNA3.1-IFITM2及靶向IFITM2的siRNA序列分别转染HEK293细胞,qPCR法和Western blot法检测IFITM2基因,mRNA的转录水平及蛋白表达水平。将EMCV按100 TCID50感染HEK293细胞,检测病毒拷贝数及病毒滴度,评价过表达及抑制表达IFITM2对EMCV增殖的影响。将EMCV按MOI=3感染过表达IFITM2的HEK293细胞,检测病毒拷贝数,评价IFITM2过表达对EMCV吸附及进入的影响。结果转染重组表达质粒后,HEK293细胞中IFITM2基因mRNA转录水平及蛋白表达水平均明显升高(P<0.001);转染siRNA序列后,两者均明显降低(P<0.001)。过表达IFITM2的HEK293细胞中的病毒拷贝数及病毒滴度明显降低(P<0.05),抑制IFITM2表达的HEK293细胞中的病毒拷贝数及病毒滴度明显升高(P<0.001)。过表达IFITM2的HEK293细胞的吸附病毒粒子数变化差异无统计学意义(P>0.05),进入病毒粒子数明显降低(P<0.01)。结论 IFITM2可抑制EMCV在HEK293细胞中的增殖,主要在病毒复制早期发挥作用。

Objective To investigate the effect of interferon-induced transmembrane protein 2(IFITM2)on proliferation of encephalomyocarditis virus(EMCV) in vitro. Methods HEK293 cells were transfected with recombinant plasmid pcDNA3. 1-IFITM2 and siRNA sequence targeting IFITM2 respectively,and determined for IFITM2 mRNA transcription level by qPCR and IFIMT2 protein expression level by Western blot. HEK293 cells were infected with 100 TCID50 of EMCV,based on which the effects of overexpression and inhibiting expression of IFITM2 on the virus proliferation were evaluated by determination of virus copy number and virus titer. The HEK293 cells in which IFITM2 was overexpressed were infected with EMCV at a MOI of 3 and evaluated for the effect of IFITM2 overexpression on adsorption and entry of EMCV by determination of virus copy number. Results Both the mRNA transcription and protein expression levels of IFITM2 in HEK293 cells transfected with recombinant plasmid pcDNA3. 1-IFITM2 increased significantly(P< 0. 001)while those transfected with siRNA sequence decreased significanthly(P< 0. 001). Both the copy number and titer of EMCV in HEK293 cells in which IFITM2 was overexpressed decreased significantly(P< 0. 05),while those in the cells in which IFITM2 expression was inhibited increased significantly(P< 0. 001). However,the number of virus particles adsorbed in HEK293 cells in which IFITM2 was overexpressed showed no significant change(P > 0. 05),while that of virus particles entered decreased significantly(P< 0. 01). Conclusion IFITM2 inhibited the proliferation of EMCV in HEK293 cells mainly at the early stage of viral replication.