【摘要】 猪氨肽酶N（porcine aminopeptidase N,pAPN）是一种锌金属蛋白酶，介导病毒与宿主细胞融合，是冠状病毒的细胞受体之一。为探究pAPN对猪流行性腹泻病毒（porcine epidemic diarrhea virus, PEDV）复制的影响，本研究从小肠组织中扩增出pAPN基因全长，通过限制性内切酶同源位点克隆到慢病毒载体中，获得携带pAPN基因的慢病毒载体PLVX-pAPN-mCMV-ZsGreen1-puro,在293T细胞中包装形成表达pAPN的慢病毒，将慢病毒感染Vero细胞，用嘌呤霉素加压筛选阳性细胞，采用有限稀释法筛选出稳定表达的Vero-pAPN细胞系，并通过测定N基因mRNA转录水平、N蛋白表达水平和TCID₅₀水平差异情况来验证该细胞系对PEDV复制的影响。结果显示，本研究构建的慢病毒能够感染Vero细胞，亚克隆筛选到的单克隆细胞株Vero-pAPN（2C5）能够稳定过表达pAPN;该细胞系能够促进PEDV复制，在12～48 h内N基因mRNA转录水平差异显著（P<0.05）、N蛋白表达水平均提高，TCID₅₀在24和48 h差异显著（P<0.05）。本研究构建了Vero-pAPN细胞系，pAPN在Vero细胞中过表达能够促进PEDV复制，该细胞系可作为疫苗高效生产和临床样本分离的候选细胞系。更多还原

【Abstract】 pAPN is a zinc-dependent metalloprotease, mediating the fusion between virus and host cell, and playing a role as the receptor of coronavirus.To explore the effect of pAPN on PEDV replication, the full-length pAPN gene was amplified from the porcine small intestinal by PCR,and was cloned into the lentiviral vector via the homologous site digested with BamHⅠ and NotⅠ to obtain the recombinant lentiviral vector PLVX-pAPN-mCMV-ZsGreen1-puro.The recombinant lentiviral vector and helper plasmids pLP1,pLP2,pLP-VSVG were co-transfected into 293T cells for lentiviral packaging.Vero cells were infected with the packaged lentivirus and the pAPN gene overexpressing cells were screened by puromycin.The stable expression of Vero-pAPN monoclonal cell line was screened by a limited dilution method, and the effect of the cell line on the replication of PEDV was determined by qPCR for N mRNA transcription level, Western blot for N protein level, and TCID₅₀.The results showed that the packaged lentivirus could infect Vero cells, and the monoclonal cell line Vero-pAPN（2C5） could stably expressed pAPN.The Vero-pAPN cell line can promote the replication of PEDV,the N gene mRNA transcription level was significantly different at 12-48h（P<0.05）,the N protein expression level increased,and the TCID₅₀ was significantly different at 24 and 48h（P<0.05）.In conclusion,the Vero-pAPN cell line was constructed in this study and it can significantly promote the replication of PEDV,which provides a candidate cell line for PEDV vaccine production and isolation.更多还原