猪流行性腹泻病毒非结构蛋白Nsp15互作宿主蛋白的筛选及鉴定

Screening and identification of host proteins interacting with the non-structural protein 15 (Nsp15) of porcine epidemic diarrhea virus

摘要:

为了筛选和鉴定与猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV)非结构蛋白15 (non-structural protein 15, Nsp15)互作的关键宿主蛋白，通过IP/pull-down beads结合质谱技术筛选、鉴定与PEDV非结构蛋白Nsp15互作的宿主蛋白，并利用免疫共沉淀(co-immunoprecipitation)、激光共聚焦验证其互作关系，最后应用Western blotting和实时荧光定量多聚核苷酸链式反应(real-time quantitative polymerase chain reaction, RT-qPCR)初步明确宿主蛋白SLC25a3与PEDV之间的作用关系。本研究成功构建了Nsp15重组真核表达载体pcDNA3.1(+)-Flag-Nsp15，并筛选出与PEDV Nsp15互作的宿主蛋白SLC25a3。免疫共沉淀及激光共聚焦结果显示PEDV Nsp15与宿主蛋白SLC25a3存在互作关系。宿主蛋白SLC25a3能够显著抑制PEDV复制且呈现剂量依赖性。本研究为进一步探究SLC25a3在抗PEDV免疫应答中的作用及机制提供了参考。

关键词:猪流行性腹泻;非结构蛋白;蛋白互作;病毒复制

Abstract:

To screen and identify the key host proteins interacting with the non-structural protein 15 (Nsp15) of porcine epidemic diarrhea virus (PEDV). The IP/pull-down assay and mass spectrometry were employed to screen and identify the host proteins interacting with Nsp15. The interaction between the host protein and Nsp15 was studied by co-immunoprecipitation and laser scanning confocal microscopy. Finally, Western blotting and RT-qPCR were employed to examine the interaction between SLC25a3 and PEDV. The recombinant eukaryotic expression vector pcDNA3.1(+)-Flag-Nsp15 was successfully constructed, and the host protein SLC25a3 interacting with PEDV Nsp15 was screened out. An interaction existed between SLC25a3 and Nsp15, and SLC25a3 significantly inhibited PEDV replication in a dose-dependent manner. SLC25a3 inhibits PEDV replication. The results of this study provide a basis for deciphering the role and mechanism of SLC25a3 in the host immune response to PEDV infection.

Key words:porcine epidemic diarrhea;non-structural proteins;protein-protein interaction;virus replication