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

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.