Construction of PCIF1 knockdown cell line and its effect on replication of porcine epidemic diarrhea virus
[Objective]The purpose of this study is to construct methyltransferase,phosphorylated C-terminal domain interacting factor 1(PCIF1)knockdown cell lines to efficiently investigate the effect and mechanism of PCIF1 on porcine epidemic diarrhea virus(PEDV)replication.[Method]In this study,a PCIF1 knockdown(PCIF1-KD)cell line was constructed using RNA interference(RNAi)technology.Initially,a PCIF1-specific targeting vector,pcDNA3.1-Double U6-mCherry-sh PCIF1,was constructed,and tranfected into African green monkey kidney cells(Vero-81)mediated by lipofectamine.The PCIF1-KD recombinant cells were then screened by geneticin,and then indentified by RT-qPCR and Western blotting.Finally,the replication capacity of the porcine epidemic diarrhea virus(PEDV)on the obtained recombinant PCIF1-KD cells was verified by RT-qPCR and Western blotting.[Result]The recombinant interference plasmid was successfully generated,as demonstrated by sequencing.The results of RT-qPCR and Western blotting showed that PCIF1 was significantly knocked down in the recombinant Vero-81 cells,and thus a stable PCIF1-KD recombinant cell line,designated as sh-△PCIF1-Vero-81,was obtained.The level of PEDV replication was markedly suppressed in sh-△PCIF1-Vero-81 cells,and this inhibiting effect was magnified from 24 to 36 hours post-infection.[Conclusion]This study constructs a PCIF1-KD Vero-81 cell line,which shows substantial inhibition of PEDV proliferation in a time-dependent manner.The results of the study could provide a powerful tool to further investigate the effect of PCIF1 on PEDV or to elucidate the molecular mechanism of how PCIF1 interacts with PEDV.
万方数据
维普
