猪流行性腹泻病毒Nsp7的亚细胞定位和对Ⅰ型干扰素应答的影响

发布时间：2018-02-24 23:13

本文关键词： 猪流行性腹泻病毒非结构蛋白 Ⅰ型干扰素天然免疫应答　出处：《畜牧兽医学报》2017年03期 　论文类型：期刊论文

【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。
[Abstract]:For the study of porcine epidemic diarrhea virus (PEDV) nonstructural protein 7 (nonstructural protein 7, Nsp7) on the cell and subcellular localization of type I interferon (IFN) response of the test using bioinformatics to predict Nsp7 gene sequence of PEDV was cloned China strains Nsp7 gene and construct the eukaryotic expression vector pCAGGS-Nsp7; the expression and distribution of Western by blot and indirect immunofluorescence assay for the detection of Nsp7 in the cell; by reporter gene method, and the effect of ELISA virus replication inhibition test evaluation of Nsp7 on type I IFN responses. PEDV analysis showed that Nsp7 gene is 249bp gene cloning and sequence is highly conserved in different PEDV strains; Western blot the results showed that Nsp7 could be efficiently expressed in transfected cells; indirect immunofluorescence assay revealed that Nsp7 mainly localized in the cytoplasm, only a small amount of the distribution in the nucleus; luciferase test Show that Nsp7 can significantly inhibit IFN- beta promoter activity, ELISA results showed that the expression of Nsp7 can significantly inhibit IFN- protein, and vesicular stomatitis virus replication inhibition test showed that Nsp7 significantly inhibited poly (I:C) type antiviral effect mediated by IFN. The results suggest that Nsp7 as a conserved protein PEDV, antagonistic type the IFN response function, to explore and reveal the PEDV escape from the host innate immune response mechanism and clinical use of vaccines to lay the foundation.

【作者单位】：河南农业大学牧医工程学院;
【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)
【分类号】：S852.651

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