Establishment of Vero cell line overexpressing pAPN gene and its effect on porcine epidemic diarrhea virus replication
10.16303/j.cnki.1005-4545.2025.02.02
- VernacularTitle:pAPN过表达Vero细胞系构建及其对PEDV复制的影响
- Author:
Qiuyong CHEN
1
;
Zhihua SUN
;
Rujing CHEN
;
Xuemin WU
;
Renjie WU
;
Jinli QIU
;
Bing HE
;
Yutao LIU
;
Longbai WANG
;
Lunjiang ZHOU
Author Information
1. 福建省农业科学院畜牧兽医研究所,福建 福州 350013
- Publication Type:Journal Article
- Keywords:
porcine epidemic diarrhea virus;
porcine aminopeptidase N;
virus proliferation;
Vero cell
- From:
Chinese Journal of Veterinary Science
2025;45(2):181-186
- CountryChina
- Language:Chinese
-
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 rep-lication,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 BamH Ⅰ and Not Ⅰ 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 12-48 h(P＜0.05),the N protein expression level increased,and the TCID50 was significantly different at 24 and 48 h(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.
