Prokaryotic Expression and Anti‑PRRSV Activity Study of Porcine IFN‑α8

doi:10.15933/j.cnki.1004?3268.2023.09.014

Journal of Henan Agricultural Sciences ›› 2023, Vol. 52 ›› Issue (9): 141-147.DOI: 10.15933/j.cnki.1004?3268.2023.09.014

• Animal Science Veterinary Medicine • Previous Articles Next Articles

Prokaryotic Expression and Anti‑PRRSV Activity Study of Porcine IFN‑α8

FANG Jianyu¹，ZHANG Qingxian¹，LANG Limin¹，XU Bin^1,2，WANG Gaili^1,2，XI Yanyan^1,2，FENG Xianming³，WANG Keling¹，LI Shaoyu^1,2

（1.Institute of Animal Husbandry and Veterinary Science，Henan Academy of Agricultural Sciences，Zhengzhou 450002，
China；2.Henan Key Laboratory of Farm Animal Breeding and Nutritional Regulation，Henan Academy of Agricultural Sciences，Zhengzhou 450002，China；3.Center for Linzhou Animal Disease Control and Prevention，Linzhou 456550，China）

Received:2023-01-05 Published:2023-09-15 Online:2023-10-11

猪IFN-α8 的原核表达及抗猪繁殖与呼吸综合征病毒活性研究

方剑玉¹，张青娴¹，郎利敏¹，徐彬^1,2，王改利^1,2，席燕燕^1,2，冯现明³，王克领¹，李绍钰^1,2

（1.河南省农业科学院畜牧兽医研究所，河南郑州 450002；2.河南省农业科学院畜禽繁育与营养调控河南省重点实验室，河南郑州 450002；3.林州市动物疫病预防控制中心，河南林州 456550）

通讯作者: 李绍钰（1965-），男，湖北麻城人，研究员，博士，主要从事动物营养调控以及抗微生物制剂研究。E-mail：lsy9617@163.com
作者简介:方剑玉（1982-），女，河南林州人，博士，主要从事动物传染病致病机制和抗病毒制剂研究。E-mail：fangjianyu2006@126.com
基金资助:
河南省科技攻关项目（222102110054）；河南省农业科学院自主创新项目（2022ZC44）

Abstract

Abstract: The aim of this study is to determine the antiviral activity of porcine interferon α8（poIFN‑α8）against porcine reproductive and respiratory syndrome virus（PRRSV）.The poIFN‑α8 gene was analyzed and synthesized，then was further cloned into the pCSMH plasmid to construct E.coli expression vector pCSMH‑IFN‑α8.The pCSMH‑IFN‑α8 was transformed into E.coli DH5α and recombinant poIFN‑α8 protein was expressed with temperature change.The recombinant protein was purified using affinity chromatography，and identified by Western blot assay.The antiviral activity of recombinant poIFN‑α8 was tested in PAM cells. And the expression of interferon stimulating genes（ISGs）induced by recombinant poIFN‑α8 protein was further detected in this experiment.The result demonstrated that inclusion expression of poIFN‑α8 protein in E.coli DH5α was achieved after the induction with temperature change，and the recombinant poIFN‑α8 could react with porcine interferon polyclonal antibody. 0. 05 ng/mL of poIFN‑α8 protein could significantly inhibit the PRRSV replication，and the antiviral effect of poIFN‑α8 was superior to porcine IFN‑α2（poIFN‑α2），porcine IFN‑α5（poIFN‑α5）.Moreover，recombinant poIFN‑α8 protein could effectively induce the expression of ISGs.To sum up，the poIFN‑α8 expressed with E.coli DH5α could significantly inhibit the PRRSV replication and activate the ISGs expression in PAM.

Key words: words：Porcine interferon α8, Escherichia coli expression, Porcine reproductive and respiratory syndrome virus, Interferon stimulating genes, Antiviral activity

摘要： 为确定猪干扰素α8（poIFN-α8）蛋白对猪繁殖与呼吸综合征病毒（PRRSV）的抗病毒作用，合成poIFN-α8 基因，克隆入pCSMH大肠杆菌表达载体，将重组表达载体转化DH5α感受态细胞，进行温度诱导表达后，收集菌体进行SDS-PAGE电泳。将表达的目的蛋白利用Ni-琼脂糖凝胶纯化柱纯化后，采用Western blot 试验检测重组poIFN-α8 的反应原性，利用猪肺泡巨噬细胞（PAM）检测0.01、0.05、0.1、1 ng/mL的重组poIFN-α8蛋白对PRRSV的抗病毒活性，并检测重组poIFN-α8蛋白对下游干扰素刺激基因（ISG）Mx1、OAS1、ISG15的诱导激活作用。结果表明，成功构建了pCSMH-IFN-α8 表达载体，实现了poIFN-α8 在大肠杆菌中的包涵体表达，且重组poIFN-α8蛋白具有良好的反应原性；0.05 ng/mL的重组poIFN-α8蛋白可显著抑制PRRSV在PAM中的复制，poIFN-α8的抗病毒效果显著优于猪干扰素α2（poIFN-α2）、猪干扰素α5（poIFN-α5），且重组poIFN-α8能有效激活ISG的表达。综上，表达的重组poIFN-α8蛋白能有效抑制PRRSV的复制，且能激活ISG的表达。

关键词: 猪干扰素α8, 大肠杆菌表达, 猪繁殖与呼吸综合征病毒, 干扰素刺激基因, 抗病毒活性

CLC Number:

S855.1

FANG Jianyu, ZHANG Qingxian, LANG Limin, XU Bin, WANG Gaili, XI Yanyan, FENG Xianming, WANG Keling, LI Shaoyu. Prokaryotic Expression and Anti‑PRRSV Activity Study of Porcine IFN‑α8[J]. Journal of Henan Agricultural Sciences, 2023, 52(9): 141-147.

方剑玉, 张青娴, 郎利敏, 徐彬, 王改利, 席燕燕, 冯现明, 王克领, 李绍钰. 猪IFN-α8 的原核表达及抗猪繁殖与呼吸综合征病毒活性研究[J]. 河南农业科学, 2023, 52(9): 141-147.

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Prokaryotic Expression and Anti‑PRRSV Activity Study of Porcine IFN‑α8

猪IFN-α8 的原核表达及抗猪繁殖与呼吸综合征病毒活性研究

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