猪Ⅲ型干扰素原核表达及其抗病毒效果研究

Prokaryotic expression of porcine typeⅢinterferon and studying on its antiviral activity

Ⅲ型干扰素(IFN-λ)具有较好的广谱抗病毒能力和免疫功能调节能力,一般在肠道等黏膜部位呈现高表达。猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)和猪德尔塔冠状病毒(porcine deltacoronavirus,PDCoV)是引起仔猪腹泻的重要肠道病原,严重困扰生猪产业健康发展。研究构建了携带猪PoIFN-λ3基因的重组表达质粒,转化大肠埃希菌Rosetta感受态细胞原核表达后,经SDS-PAGE和Western blotting检测分析,重组PoIFN-λ3蛋白成功表达,大小约为27 ku,且以包涵体形式存在。变性、纯化、复性后的PoIFN-λ3重组蛋白刺激IPEC-J2细胞,ISG-15、OAS1、Mx-1、IFIT1、IFIT3、IFITM1和IFITM3等干扰素刺激基因(ISGs)表达均显著上调,且在12 h达到峰值(P<0.001)。IPEC-J2细胞分别感染PEDV和PDCoV病毒时,用PoIFN-λ3重组蛋白预处理、同时处理和感染后处理这3种方式处理后观察病毒增殖情况,与对照组相比,PEDV和PDCoV病毒拷贝数均显著下降(P<0.05)。上述结果表明,变性、纯化、复性后的PoIFN-λ3重组原核表达蛋白具有较好的生物活性,不仅可以诱导IPEC-J2细胞免疫刺激基因表达上调,在体外也可以抑制PEDV和PDCoV在IPEC-J2细胞中的复制,为防治仔猪病毒性腹泻提供了基础。

Normally,type III interferon(IFN-λ)was highly expressed in intestinal mucosa and other mucosal systems,with relatively stronger broad-spectrum antiviral ability and immune regulating ability.Porcine epidemic diarrhea virus(PEDV)and porcine deltacorona virus(PDCoV)were two major intestinal pathogens causing diarrhea in piglets,which hindered the swine industry severely.In this study,the recombinant plasmid containing PoIFN-λ3 fragment was constructed.The recombinant plasmid was then transformed into E.coli Rosaetta cells for recombinant expression.Analyzed by SDS-PAGE and western blotting,the results showed that the recombinant PoIFN-λ3(27 ku)was successfully expressed in the inclusion bodies of E.coli cells.After denaturation,purification and renaturation,the active PoIFN-λ3 recombinant protein was obtained and was used to treat IPEC-J2 cells.Challenged by recombinant PoIFN-λ3,the immune-stimulating genes(ISGs),such as ISG-15,OAS1,Mx-1,IFIT1,IFITM1 and IFITM3 were all up-regulated significantly and reached to the peak(P<0.001)at 12 h post challenge.For PEDV and PDCoV infection in IPEC-J2,the virus replication was detected after pretreatment,simultaneous treatment and post-infection treatment of recombinant PoIFN-λ3.Compared with the control,the viral copy numbers of both PEDV and PDCoV were decreased significantly(P<0.05)after treatment of recombinant PoIFN-λ3.The above results indicated that the purified and renatured PoIFN-λ3 recombinant protein could have good biological activity.The recombinant PoIFN-λ3 can induce high expression of different ISGs in IPEC-J2,and can also inhibit PEDV and PDCoV replication in the host cells.In summary,our study provided a basis for preventing and treating viral diarrhea in piglets.