Porcine deltacoronavirus (PDCoV) is a newly identiifed enteropathogenic coronavirus, causing diarrhea in suckling piglets. The nonstructural protein 5 (Nsp5) encoded by coronavirus is a 3C-like protease, and plays a key role in the process of viral polyproteins and immune regulation. In this study, the Nsp5 gene of PDCoV CHN-HN-2014 strain was amplified and subcloned into prokaryotic expression vector pET-28a(+), generating the recombinant expressing plasmid. After induction with IPTG, the fusion protein Nsp5-His was efifciently expressed in E.coli BL21 (DE3) in soluble form. The recombinant protein was further puriifed with Ni-NTA afifnity column and its protease activity was detected by fluorescence resonance energy transfer (FRET) method. The results showed that the puriifed recombinant protein efifciently cut the chemosynthetic 12 peptide that contained the N-terminal cleavage site of PDCoV Nsp5, indicating that Nsp5 possessed protease activity. The progress in cloning, expression and analysis of protease activity of Nsp5 will lay the foundation for the future functional research of PDCoV.%猪δ冠状病毒（Porcine deltacoronavirus, PDCoV）是一种新发现的肠道冠状病毒，可引起仔猪的腹泻。冠状病毒编码的非结构蛋白Nsp5是一种3C样蛋白酶，在病毒多聚前体蛋白加工以及免疫调节中发挥重要作用。本研究以PDCoV CHN-HN-2014株为模板，通过RT-PCR扩增Nsp5基因并将其克隆到原核表达载体pET-28a（+）中，构建了重组表达质粒。将重组质粒转化到大肠杆菌BL21（DE3），经IPTG诱导后SDS-PAGE检测，结果表明Nsp5可在大肠杆菌中高效、可溶性表达。采用镍亲和层析对表达的Nsp5蛋白进行纯化，并通过荧光共振能量转移方法检测了其蛋白酶活性，结果表明纯化的Nsp5蛋白能够有效切割含有PDCoV Nsp5氨基端自切割位点的十二肽底物，证实体外表达的Nsp5重组蛋白具有良好的蛋白酶切割活性。猪δ冠状病毒Nsp5蛋白的克隆、表达及其蛋白酶活性检测，为深入研究该蛋白的功能奠定了基础。
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