猪丁型冠状病毒S1-CTD相互作用宿主蛋白的筛选和鉴定

doi:10.11843/j.issn.0366-6964.2022.07.022

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (7): 2260-2267.doi: 10.11843/j.issn.0366-6964.2022.07.022

猪丁型冠状病毒S1-CTD相互作用宿主蛋白的筛选和鉴定

汤荣锋, 范前进, 郭龙军, 张鑫, 石达, 时洪艳, 陈建飞*, 冯力*

中国农业科学院哈尔滨兽医研究所, 兽医生物技术国家重点实验室/猪消化道传染病创新团队, 哈尔滨 150069

收稿日期:2021-10-26 出版日期:2022-07-23 发布日期:2022-07-23
通讯作者: 陈建飞,主要从事兽医微生物及其分子生物学研究,E-mail:chenjianfei@caas.cn;冯力,主要从事动物疫苗与分子免疫学研究,E-mail:fengli@caas.cn
作者简介:汤荣锋(1997-),男,硕士,主要从事猪肠道冠状病毒的研究,E-mail:souprf@163.com
基金资助:
黑龙江省科学基金项目(TD2020C002)；兽医生物技术国家重点实验室自主研究课题(SKLVBP202105)

Screening and Identification of Host Proteins Interacting with PDCoV S1-CTD

TANG Rongfeng, FAN Qianjin, GUO Longjun, ZHANG Xin, SHI Da, SHI Hongyan, CHEN Jianfei*, FENG Li*

Swine Digestive System Infectious Diseases Division, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, State Key Laboratory of Veterinary Biotechnology, Harbin 150069, China

Received:2021-10-26 Online:2022-07-23 Published:2022-07-23

摘要/Abstract

摘要： 猪丁型冠状病毒纤突蛋白S1亚基C端结合域(S1-CTD)是诱导中和抗体产生的主要区域，为研究与其相互作用的宿主蛋白，采用HEK-293T真核表达系统表达并纯化了S1-CTD，提取了猪回肠上皮细胞膜蛋白，通过免疫共沉淀筛选了可能与S1-CTD相互作用的蛋白并进行质谱分析，发现32个疑似相互作用的宿主蛋白。构建其中可能与S1-CTD互作的蛋白KIF1 binding protein (KIFBP)的真核表达质粒，通过免疫共沉淀和激光共聚焦验证上述宿主蛋白与S1-CTD是否存在相互作用，结果表明KIFBP和S1-CTD之间存在相互作用，共同转染时在细胞质共定位。进一步研究表明，过表达KIFBP能够有效降低病毒RNA水平和病毒滴度，其中mRNA水平降低了约70%，病毒滴度降低了10^1.6TCID₅₀。综上，本研究筛选并鉴定出一种与PDCoV S1-CTD相互作用的宿主蛋白KIFBP，为了解PDCoV的致病机制提供了理论基础。

关键词: 猪丁型冠状病毒, S1-CTD蛋白, 相互作用, KIFBP

Abstract: The C-terminal domain (CTD) of porcine deltacoronavirus S1 subunit is the main region which induces the neutralizing antibody. S1-CTD was expressed by HEK-293T eukaryotic expression system and purified, and porcine ileal epithelium cells membrane proteins were extracted to investigate porcine host proteins that interact with it. Thirty-two suspected interacting host proteins were obtained by co-inmunprecipitation (Co-IP) and mass spectrometry. Eukaryotic expression plasmid of KIF1 binding protein (KIFBP) was constructed, and the interaction between KIFBP and S1-CTD was identified by Co-IP and laser confocal microscopy. All results proved that KIFBP interacted with S1-CTD and co-located in cytoplasm. Further research indicated that overexpression of KIFBP could effectively reduce the viral mRNA level and the viral titer in which the mRNA level decreased by about 70%, and the viral titer decreased by 10^1.6TCID₅₀. In conclusion, a host protein KIFBP interacting with PDCoV S1-CTD was screened and identified in this study which provides a theoretical basis for understanding the pathogenesis of PDCoV.

Key words: porcine deltacoronavirus, S1-CTD protein, interaction, KIF1 binding protein

中图分类号:

S852.659.6

汤荣锋, 范前进, 郭龙军, 张鑫, 石达, 时洪艳, 陈建飞, 冯力. 猪丁型冠状病毒S1-CTD相互作用宿主蛋白的筛选和鉴定[J]. 畜牧兽医学报, 2022, 53(7): 2260-2267.

TANG Rongfeng, FAN Qianjin, GUO Longjun, ZHANG Xin, SHI Da, SHI Hongyan, CHEN Jianfei, FENG Li. Screening and Identification of Host Proteins Interacting with PDCoV S1-CTD[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2260-2267.

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猪丁型冠状病毒S1-CTD相互作用宿主蛋白的筛选和鉴定

Screening and Identification of Host Proteins Interacting with PDCoV S1-CTD

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