猪流行性腹泻病毒E蛋白诱发细胞未折叠反应的分子机制

Molecular mechanism of unfolding response induced by porcine epidemic diarrhea virus E protein

为阐明猪流行性腹泻病毒(PEDV) E蛋白诱导Vero-E6细胞内质网应激分子机制,采用RT-PCR方法扩增得到E蛋白基因,经XhoⅠ和BamHⅠ酶切后与同样酶切的p-EGFP-N1载体使用T4 DNA连接酶进行连接,成功构建了表达载体,与pCDNA 3.1-GRP78共转染Vero-E6细胞,通过激光共聚焦试验和Western blot验证E蛋白的细胞定位,并揭示其激活细胞内质网应激的分子机制。结果表明,通过共聚焦荧光显微镜证实E蛋白定位在细胞中,Western blot方法验证E蛋白可以上调Vero-E6细胞GRP78蛋白表达水平,诱导Vero细胞内质网应激。进一步采用Western blot验证E蛋白通过PERK-eIF2α信号通路激活未折叠蛋白反应。本研究为探究PEDV致病机制提供了新的思路。

To fully illustrate the molecular mechanism of how the porcine epidemic diarrhea virus(PEDV) E induces unfolded protein response(UPR), the E gene was obtained through RT-PCR, and the acquired gene fragment and the p-EGFP-N1 vector were digested by XhoⅠ and BamHⅠ. Then, the E gene and p-EGFP-N1 were ligated by T4 DNA enzyme to construct a p-EGFP-N1-E eukaryotic expression vector. p-EGFP-N1-E and p-EGFP-N1 were transfected into Vero-E6 cells with pCDNA 3.1-GRP78 plasmids to confirm the localization of the E protein, the molecular mechanism of the E protein inducing endoplasmic reticulum stress(ERS), and the molecular mechanism of the E protein inducing unfolded protein response(UPR). Western blot confirmed the higher expression of pCDNA 3.1-GRP78, compared with NC, which suggested that the E protein induced ERS in the Vero-E6 cells. Further research confirmed that p-EGFP-N1-E induced UPR via the PERK-eIF2α pathway. This study provided a new direction for research on the molecular mechanism of PEDV.