伪狂犬病病毒在NF-κB家族p65基因敲除细胞系的复制规律研究

doi:10.16431/j.cnki.1671-7236.2021.01.009

Abstract

Abstract: The aim of this study was to find out the replication of Pseudorabies virus (PRV) in cell lines with NF-κB family the p65 gene knocked out.The 3D4/21 cell lines with p65 gene stable knocked out were constructed by site-specific gene modification techniques CRISPR/Cas9.The recombinant plasmid p65-sgRNA was constructed and transfected into HEK293T/17 cells.Lentivirus was collected and infect 3D4/21 cells.The polyclonal cell lines was gained by puromycin screening.The knockout efficiency was detected by T7 nuclease digestion.The stable cell line of 3D4/21-p65^-/- was obtained by finite dilution method.The effect of p65 gene knockout on cell proliferation in 3D4/21 cell lines was detected by CCK-8 kit.The difference of virus proliferation in 3D4/21 and 3D4/21-p65^-/- was detected by flow cytometry.The mRNA expression level of PRV gB,TK,IL-1β and IL-6 gene were detected by Real-time quantitative PCR after infecting PRV-QXX in 3D4/21 and 3D4/21-p65^-/- cells.The protein expression of PRV gB and gE were detected by Western blotting after infecting PRV-QXX in 3D4/21 and 3D4/21-p65^-/- cells.The titration of the progeny PRV-QXX produced by infecting PRV-QXX in 3D4/21 and 3D4/21-p65^-/- cells was measured by traditional plaque formation assay and developed median tissue culture infective dose(TCID₅₀)methods.The results showed that the gene editing efficiency of sgRNA2 and sgRNA3 was higher.The stable expression cell lines with p65 gene knockout were obtained by cloning and culturing.CCK-8 kit test was used to detect cell viability,result showed that p65 gene knockout had no effect on it.The result of flow cytometry detection at the same time point showed that the proliferation of PRV-GFP in 3D4/21-p65^-/- cells was significantly higher than that in control cells.The result of Real-time quantitative PCR was exhibit that in 3D4/21 cells,p65 gene knockout promoted the mRNA expression of PRV gB and TK gene,but IL-1β and IL-6 genes were inhibited.Western blotting was used to demonstrate that p65 gene knockout promoted the expression of PRV gB and gE protein in 3D4/21 cells.The virus titer result gene showed that the replication of PRV-QXX was significantly higher than that of control cells in 3D4/21-p65^-/- at the same time.The above results suggested that p65 gene knockout promoted PRV replication in 3D4/21 cells.

Key words: CRISPR/Cas9; NF-κB; porcine; Pseudorabies virus (PRV)

CLC Number:

Q812

CHANG Wenru, DUAN Lifang, YANG Le, MA Yingxian, WANG Qi, ZHAI Yunyun, ZHOU Luyu, ZHANG Shuang, MING Shengli, YANG Guoyu, WANG Jiang, CHU Beibei. Study on Replication Patterns of Pseudorabies Virus in Cell Lines with NF-κB Family p65 Gene Knocked Out[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(1): 83-92.

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Study on Replication Patterns of Pseudorabies Virus in Cell Lines with NF-κB Family p65 Gene Knocked Out

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