猪流行性腹泻病毒nsp1对Ⅰ型干扰素应答的影响

摘要

猪流行性腹泻病毒(PEDV)能抑制宿主Ⅰ型干扰素及其诱导的细胞抗病毒免疫应答,但是PEDV抑制Ⅰ型干扰素应答的分子机制尚不明了,尤其是PEDV非结构蛋白(Nonstructural proteins,nsps)在Ⅰ型干扰素应答中的调控作用研究不多.为研究PEDV非结构蛋白1(nspl)对细胞Ⅰ型干扰素应答的影响,构建了真核表达载体pCAGGS-nsp1,采用Western blotting和间接免疫荧光试验确定nsp1在细胞中的表达.通过报告基因法、ELISA以及病毒复制抑制试验评估nspl对Ⅰ型IFN的影响.结果显示,nspl在转染细胞和病毒感染细胞中均高效表达.双荧光报告基因试验结果表明,nspl能显著抑制IFN-p启动子活性,且具有剂量依赖性.ELISA结果显示,nspl能显著抑制IFN-β蛋白的表达.水泡性口炎病毒(VSV)复制抑制试验结果显示,nsp1明显抑制poly(I:C)介导的Ⅰ型IFN的抗病毒作用.结果提示,nsp1作为PEDV的保守蛋白,具有拮抗Ⅰ型干扰素启动子活性和应答的功能,为揭示PEDV逃逸宿主天然免疫应答的机制和研发新型高效抗PEDV疫苗奠定基础.%Porcine epidemic diarrhea virus (PEDV) inhibits the host type Ⅰ interferon and cellular antiviral response,but its inhibition mechanism is unclear,and the roles of PEDV nonstructural proteins in regulating type Ⅰ interferon responses have been seldom studied.To study the effect ofnspl on type Ⅰ interferon response,nspl gene was cloned into a eukaryotic expression vector pCAGGS.The expression of nspl in transfected cells was determined by Western blot and indirect immunofluorescence assay.The effects of nspl on the induction of type Ⅰ interferon were evaluated by dual luciferase reporter gene assay,ELISA and VSV bioassay.Western blot and indirect immunofluorescence assay showed that nspl was highly expressed in transfected cells and PEDV-infected cells.Dual luciferase reporter gene assay results indicated that nspl strongly inhibited the IFN-β promoter activity,and the inhibitory effect was nspl dose-dependent.ELISA results showed that nsp 1 significantly inhibited the expression of IFN-β in protein level.And VSV replication-inhibition bioassay revealed that nspl significantly inhibited type Ⅰ IFN antiviral activities induced by poly(I:C).Our results implied that nspl was a highly conserved protein of PEDV and exhibited antagonistic function on interferon promoter activity.The results have laid a foundation for further understanding the immune evasion mechanism of PEDV and for developing new effective vaccine against PEDV.