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  

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

作　　者：方剑玉[1] 张青娴[1] 郎利敏[1] 徐彬[1,2] 王改利 席燕燕 冯现明 王克领[1] 李绍钰[1,2] 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)

机构地区：[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

出　　处：《河南农业科学》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)。

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.

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 

分 类 号：S855.1[农业科学—临床兽医学]