Construction of PK-15 cell line with LTA4H gene knockout and its impact on the replication of foot-and-mouth disease virus

Abstract:

【Objective】 A LTA4H-deleted porcine kidney cell line (PK-15) was constructed by CRISPR/Cas9 to study the effect of LTA4H on the replication of foot-and-mouth disease virus (FMDV), with a view to providing a theoretical basis for revealing the functions of LTA4H and the mechanism of regulating virus replication. 【Methods】 Two small guide RNAs (sgRNAs) targeting porcine LTA4H were designed and integrated into the vector pX459-puro-MCS, respectively. The recombinant plasmid was transfected into PK-15 cells, which were then cultured in the medium supplemented with puromycin, and the monoclonal cells were selected by limiting dilution method. Western blotting and sequencing were performed to detect LTA4H knockout, and LTA4H-deleted cells were obtained. Furthermore, Western blotting, RT-qPCR, and virus titer assay were employed to examine FMDV replication and protein expression after LTA4H knockout. 【Results】 Compared with the wild type cells, the LTA4H-deleted PK-15 cells exhibited inhibited FMDV replication. 【Conclusion】 This study successfully constructed a PK-15 cell line with LTA4H gene knockout, demonstrating that LTA4H can promote the replication of FMDV, and the results provided a theoretical basis for the subsequent research on the function of LTA4H.