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。
The aim of this study is to determine the antiviral activity of porcine interferonα8(polFN-α8)against porcine reproductive and respiratory syndrome virus(PRRSV).The polFN-a8 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 DH5αand 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 polFN-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 DH5αwas achieved afer the induction with temperature change,and the recombinant polFN-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 DH5αcould significantly inhibit the PRRSV replication and activate the ISGs expression in PAM.
作者
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
FANG Jianyu;ZHANG Qingxian;LANG Limin;XU Bin;WANG Gaili;XI Yanyan;FENG Xianming;WANG Keling;LI Shaoyu(Institute of Animal Husbandry and Veterinary Science,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;Henan Key Laboratory of Farm Animal Breeding and Nutritional Regulation,Henan Academy of Agricultural Sciences,Zhengzhou 450002,China;Center for Linzhou Animal Disease Control and Prevention,Linzhou 456550,China)
出处
《河南农业科学》
北大核心
2023年第9期141-147,共7页
Journal of Henan Agricultural Sciences
基金
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)。
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
Porcine interferon a8
Escherichia coli expression
Porcine reproductive and respiratory syndrome virus
Interferon stimulating genes
Antiviral activity
