猪流行性腹泻病毒在稳定表达猪ACE2基因的IPEC-J2细胞中的增殖研究

Study on Proliferation of a PEDV Strain on IPEC-J2 Cells Stably Expressing Porcine ACE2

为获得稳定表达猪源血管紧张素转换酶2(ACE2)的细胞株,用以增殖猪流行性腹泻病毒(PEDV)。构建了稳定表达猪源ACE2的猪小肠上皮细胞系(IPEC-J2),并对PEDV在该细胞上的增殖进行了分析。将重组表达质粒pCMV3-porcine-ACE2-GFP转染至IPEC-J2细胞后,经潮霉素B筛选和单克隆细胞培养,获得IPEC-J2-ACE2细胞系。通过荧光定量RT-PCR和Western blot对单克隆细胞中ACE2基因的mRNA转录和蛋白表达水平进行鉴定。以PEDV-WH株感染IPEC-J2-ACE2细胞,通过间接免疫荧光试验和荧光定量RT-PCR检测发现,PEDV-WH株在IPEC-J2-ACE2细胞中比在IPEC-J2细胞中的增殖能力高。

In order to get a cell line susceptive to porcine epidemic diarrhea virus(PEDV)infection,we used eukaryotic expression of porcine angiotensin-converting enzyme 2(ACE2)in intestinal porcine epithelial cell line J2(IPEC-J2).And the replication of PEDV in IPEC-J2was analysed.Firstly,the recombinant plasmid pCMV3-porcine-ACE2-GFP was transfected to IPEC-J2cells.In order to obtain the monoclonal cell line IPEC-J2-ACE2,the transfected cells were selected with hygromycin B and using limited dilution method to get.Then,we used real-time PCR and Western blot methods to analyze the mRNA and protein expression levels of ACE2.The results displayed that porcine ACE2protein was exactly and stably expressed in IPEC-J2-ACE2cells.Moreover,we used PEDV-WH strain to infect IPEC-J2-ACE2cells,then the results of indirect immunofluorescence assay and real-time PCR indicated that PEDV can replicated in IPEC-J2-ACE2cells.Comparing to the control group that PEDV infected IPEC-J2cells,the relative mRNA expression of PEDV N gene in PEDV infected IPEC-J2-ACE2cells is higher than that in control group.It showed that PEDV is more susceptible to IPEC-J2-ACE2cells.In conclusion,IPEC-J2-ACE2cell has been successfully got,which is not only possibly useful for study the replication and vaccine of PEDV,but also lays the foundation for the research of pathogenic mechanism of PEDV.