The Effect Of Nuclear Factor Kappa B Family P65 Gene Knockout On The Proliferation Of Pseudorabies Virus

Pseudorabies is an acute,violent and highly contact animal infectious disease caused by the pseudorabies virus,which has caused serious economic losses to the pig industry.There are no effective treatment drugs,mainly through vaccination to prevent large-scale outbreaks of pseudorabies,so the production of vaccines is very important.At present,in the production process of vaccines,cells are mainly used to cultivate a large number of viruses,so it is particularly important to obtain high-titer pseudorabies virus.In order to increase the amount of virus proliferation in cultured cells,this experiment explored whether it can promote the rapid and massive proliferation of viruses in cells by knocking out the NF-κB family P65 genes involved in the innate immune pathway.Provide a theoretical basis for obtaining high-titer virus in vaccine production.In order to study the role of NF-κB P65 gene in the proliferation of porcine pseudorabies virus,this experiment successfully constructed the 3D4/21-P65-/-cell line through lentivirus-mediated CRISPR/Cas9 technology,and detected the PRV in The proliferation of them.The results of the study show that knocking out the NF-κB P65 subunit promotes PRV infection.In this study,we first designed a single stranded RNA of P65 gene and connected it to LentiCRISPRv2 vector.Next,we used lentiviral packaging plasmid in order to make P65-sgRNA recombinant vector integrated into HEK293T/17 genome,and we used the lentiviral solution to infect 3D4/21 cells.After puromycin screening,P65 gene knockout polyclonal cells were obtained,and the knockout efficiency of P65 gene was obtained by T7 digestion.The 3D4/21-P65-/-cell line was obtained by limited dilution method and identified by DNA sequencing.Next,PRV-GFP virus was inoculated into 3D4/21 and 3D4/21-P65-/-cells,and the difference of GFP fluorescence intensity was detected by flow cytometry;Secondly,PRV-QXX virus was inoculated into 3D4/21 and 3D4/21-P65-/-cells respectively,and then the effect of P65 gene knockout on PRV-QXX titer was detected;what’s more,Western blotting was used to detect the difference of PRV gE and gB protein expression,Finally,the transcription levels of PRV gB and TK,IL-1β and IL-6 genes were detected by Real time fluorescent quantitative PCR.The results showed that P65-sgRNA recombinant vector was successfully constructed,and P65-sgRNA was integrated into 3D4/21 cell genome by lentivirus.A 3D4/21-P65-/-cell line was successfully obtained by T7 enzyme digestion,limited dilution and genome sequencing.And then through fluorescence observation and flow cytometry,it was found that the fluorescence intensity of GFP in 3D4/21-P65-/-cells was significantly higher than that in 3D4/21 normal cells.Besides,western blotting results showed that the expression of PRV gE and gB in 3D4/21-P65-/-cells was higher than that in 3D4/21 normal cells.Finally,when we compared the results of the two groups by real-time fluorescent quantitative PCR,we found that the transcription levels of gB and TK genes of PRV in 3D4/21-P65-/-cells were significantly higher than those in 3D4/21 normal cells;However,the mRNA expression levels of IL-1βand IL-6 in 3D4/21-P65-/-cells were significantly lower than those in 3D4/21 normal cells.These results suggest that P65 gene knockout is beneficial to PRV replication,which may be achieved by inhibiting the expression of IL-1β and IL-6.This study can provide a theoretical basis for obtaining high-titer pseudorabies virus in vaccine production.