Contruction Of TBK1 Knockout Marc-145 Cell Line And Preliminary Evaluation Of PRRSV Reproduction Status

Porcine reproductive and respiratory syndrome seriously damages the healthy development of the world pig industry.The annual investment in vaccines to prevent and control the disease is enormous.At present,the PRRS vaccine is mostly produced by Marc-145 cells,and the virus titer directly affects the effectiveness of vaccine and production cost.However,Marc-145 cells have the problem of low titer of reproductive virus.TBK1 gene is an important hub of host antiviral natural immune signal pathway,which has an important influence on the production of cell interferon.In this study,the TBK1 knockout Marc-145 cell line was constructed using CR1SPR/Cas9 technology to increase the titer of the virus.First,this study designed two pairs of sgRNAs for the homologous regions of multiple transcripts of the TBK1 gene.Using pSpCas9(BB)-2A-puro(PX459)plasmid as a vector,the double-stranded sgRNA after annealing was ligated with the PX459 plasmid linearized by Bbsl.Finally,we successfully constructed the PX459+sgRNA recombinant plasmid.Then,the recombinant plasmid was transfected into well-grown Marc-145 cells using liposome 2000 transfection reagent,and polyclonal cells were obtained by puromycin screening.The polyclonal cells were then diluted to a 96-well cell plate,and after 5 days,they were observed under a microscope every day to select monoclonal cells.After a round of Touchdown PCR identification,two rounds of Touchdown PCR amplification was sent to sequencing identification.Finally,we screened two TBK1 knockout Marc-145 cells,numbered 1-3-A,2-2-18.Furthermore,in this study,we selected 3 strains of highly pathogenic porcine reproductive and respiratory syndrome(JXA1-R,JXja-15,JXA1-E6,)and 2 strains of NADC30-Iike(HNjz-15,20160802-22)and VR2332 strains and 2 strains of European strain(180900-5,p073-3),the titers of viruses were determined in two knockout cell lines and original cell lines,respectively.As a result,except for the two European strains(180900-5,p073-3),no cytopathic effect was observed in all the cells,and the titers of the other six strains on the two knockout cells were significantly increased.The increase range is 3-12 times.In summary,this study provides new ideas for increasing the proliferation titer of PRRS virus,and the obtained 1-3-A and 2-2-18 cell lines have potential for vaccine production.