摘要
为研究猪流行性腹泻病毒(PEDV)非结构蛋白7(nonstructural protein 7,Nsp7)的亚细胞定位和对细胞Ⅰ型干扰素(IFN)应答的影响,本试验利用生物信息学预测Nsp7基因序列,克隆了PEDV中国流行毒株Nsp7基因并构建真核表达载体pCAGGS-Nsp7;采用Western blot和间接免疫荧光试验检测Nsp7在细胞中的表达和分布;通过报告基因法、ELISA以及病毒复制抑制试验评估Nsp7对Ⅰ型IFN应答的影响。基因克隆和序列分析结果显示PEDV Nsp7基因大小为249bp,在不同PEDV毒株间高度保守;Western blot结果显示,Nsp7能够在转染细胞中高效表达;间接免疫荧光试验显示Nsp7主要定位在细胞质,仅少量分布在细胞核;双荧光报告基因试验表明Nsp7能显著抑制IFN-β启动子活性,ELISA结果显示Nsp7能显著抑制IFN-β蛋白的表达,同时水疱性口炎病毒复制抑制试验显示Nsp7明显抑制poly(I:C)介导的Ⅰ型IFN的抗病毒作用。结果提示,Nsp7作为PEDV的保守蛋白,具有拮抗Ⅰ型IFN应答的功能,为探讨和揭示PEDV逃逸宿主天然免疫应答的机制及指导临床疫苗使用奠定基础。
To investigate subcellular localization and effect on typeⅠinterferon（IFN）response of nonstructural protein 7（Nsp7）of porcine epidemic diarrhea virus（PEDV）,the Nsp7 gene sequence was predicted by bioinformatics,the Nsp7 gene of Chinese PEDV epidemic strain was cloned and inserted into the eukaryotic expression vector pCAGGS.The expression and subcellular localization of Nsp7 in transfected cells were determined by Western blot and indirect immunofluorescence assay respectively.The effect of Nsp7 on typeⅠIFN response was evaluated by dual luciferase reporter gene assay,ELISA and virus replication-inhibition bioassay,respectively.Gene cloning and sequence analysis results showed that PEDV Nsp7 gene was 249 bp in length,and it was highly conserved among different PEDV strains.Western blot and indirect immunofluorescence assay showed that Nsp7 was highly expressed and localized mainly in cytoplasm.Dual luciferase reporter gene assay indicated that Nsp7 strongly inhibited the IFN-βpromoter activity.ELISA results showed that Nsp7 could significantly inhibit the expression of IFN-βin the protein level.VSV replication assay revealed that Nsp7 significantly inhibited typeⅠIFN antiviral activity induced by poly（I：C）.Our results implied that Nsp7 was a highly conserved protein of PEDV and exhibited antagonistic function on type Ⅰ IFN response.The results have laid a foundation for exploring the innate immunity evasion mechanism of PEDV and guiding the clinical application of vaccines.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2017年第3期501-507,共7页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
河南省高校创新团队支持计划(14IRTSTHN015)
河南省高等学校重点科研项目(16A230002)
