Abstract: The aim of this study was to construct a PK 15 cell line stably expressing the E165R protein of African swine fever virus (ASFV). ASFV E165R gene was ligated to p3×FLAG-CMV-10 vector by PCR amplification, and then ligated to lentivirus vector by homologous recombination to obtain recombinant plasmid V2-FLAG-E165R. Lentivirus particles were obtained by lentivirus packaging in HEK 293T cells and transduced into PK 15 cells. The polyclonal cells obtained after primary screening with puromycin were then screened by endpoint dilution method to obtain a monoclonal PK 15 cell line stably expressing E165R protein. The constructed PK 15 cell line was identified by PCR amplification, Western blot and indirect immunofluorescence, and the mRNA expression levels of NF-κB, inflammation-related genes IL-6, IL-8 and TNF-α were further detected by RT-qPCR. The results showed that a PK 15 stable cell line overexpressing ASFV E165R protein was successfully constructed. Compared with the control cell line, E165R protein significantly enhanced the gene expression of NF-κB as well as inflammation-related genes P65、IKBa、IL-6, IL-8 and TNF-ɑ. These results will provide biological materials for further research on the effects of ASFV E165R on innate immune signaling pathway.

Key words: African swine fever virus, E165R protein, cell lines, NF-κB