HMCES蛋白促进猪流行性腹泻病毒在Vero细胞中的复制

HMCES protein promotes the replication of porcine epidemic diarrhea virus in Vero cells

摘要:

【目的】鉴定能够调控猪流行性腹泻病毒(porcine epidemic diarrhea virus，PEDV)复制的关键宿主蛋白。【方法】利用LC-MS/MS技术结合串联质谱标签(tandem mass tag，TMT)，分析PEDV感染Vero细胞36 h后和未感染组的蛋白组学差异。鉴定筛选了114个显著差异表达蛋白，其中宿主胚胎干细胞特异性5-羟甲基胞嘧啶结合蛋白(5-hydroxymethylcytosine binding,ES cell-specific protein，HMCES)显著上调。进一步构建HMCES真核表达质粒，通过蛋白免疫印迹和实时荧光定量PCR检测过表达HMCES对PEDV复制的影响；合成针对HMCES基因的特异性siRNA，利用Western blotting和RT-qPCR检测siRNA对HMCES表达的干扰效果及HMCES被干扰后对PEDV复制的影响。【结果】过表达HMCES能显著促进PEDV在Vero细胞中复制，并且复制水平随着HMCES的剂量递增呈现剂量依赖式增加；siRNA-341下调内源性HMCES表达进而抑制PEDV复制。【结论】HMCES促进PEDV复制，本研究为进一步探究HMCES在抗PEDV免疫应答中的作用及机制提供了参考依据。

Abstract:

[Objective] To identify the key host protein that can regulate the replication of porcine epidemic diarrhea virus (PEDV). [Methods] We usedliquid chromatography with tandem mass spectrometry (LC-MS/MS) technology combined with tandem mass tag (TMT) to analyze the proteomic differences between PEDV-infected Vero cells and the uninfected group at 36 h post inoculation. A total of 114 significantly differentially expressed proteins were identified, of which 5-hydroxymethylcytosine binding, ES cell-specific protein (HMCES) was significantly up-regulated. The eukaryotic expression plasmid of HMCES was further constructed, and the effect of HMCES overexpression on PEDV replication was examined by Western blotting and real-time fluorescent quantitative PCR. The specific siRNA against HMCES gene was synthesized, and Western blotting and RT-qPCR were employed to detect the effect of siRNA on HMCES expression and the effect of interfered HMCES on PEDV replication. [Results] Overexpression of HMCES significantly promoted PEDV replication in Vero cells, and the level of replication increased in a dose-dependent manner. siRNA-341 down-regulated the expression of endogenous HMCES to inhibit PEDV replication. [Conclusion] HMCES promotes PEDV replication in Vero cells. This study provides a reference for further exploring the role and mechanism of HMCES in the anti-PEDV immune response.