生物工程学报2024,Vol.40Issue(12) :4533-4545.DOI:10.13345/j.cjb.240083

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

孙金磊 于瑞明 张莉萍 张中旺 王永录 潘丽 张全伟 刘新生
生物工程学报2024,Vol.40Issue(12) :4533-4545.DOI:10.13345/j.cjb.240083

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

孙金磊 1于瑞明 2张莉萍 2张中旺 2王永录 2潘丽 2张全伟 1刘新生2
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作者信息

  • 1. 甘肃农业大学 生命科学技术学院,甘肃 兰州 730070
  • 2. 中国农业科学院兰州兽医研究所 动物疫病防控全国重点实验室,甘肃 兰州 730000
  • 折叠

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.

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.

关键词

猪流行性腹泻/非结构蛋白/蛋白互作/病毒复制

Key words

porcine epidemic diarrhea/non-structural proteins/protein-protein interaction/virus replication

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出版年

2024
生物工程学报
中国科学院微生物研究所 中国微生物学会

生物工程学报

CSTPCDCSCD北大核心
影响因子:0.641
ISSN:1000-3061
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