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

试验旨在研究伪狂犬病病毒（PRV）在NF-κB家族p65基因敲除细胞系中的复制规律。利用慢病毒介导的CRISPR/Cas9基因定点修饰技术构建猪肺泡巨噬细胞（3D4/21）p65基因稳定敲除细胞系。通过构建p65-sgRNA重组质粒，转染至HEK293T/17细胞，收取慢病毒，感染3D4/21细胞后利用嘌呤霉素筛选获得多克隆细胞系，T7核酸酶检测敲除效率，再通过有限稀释法获得3D4/21-p65^-/-的稳定细胞系。CCK-8试剂盒检测3D4/21细胞中敲除p65基因后对细胞增殖的影响；流式细胞术检测PRV-GFP感染3D4/21及3D4/21-p65^-/-细胞后病毒增殖的差异；实时定量PCR检测PRV感染3D4/21及3D4/21-p65^-/-细胞后PRV gB、TK基因mRNA表达水平及PRV感染细胞诱导的IL-1β和IL-6基因mRNA水平表达的变化；Western blotting检测PRV-QXX感染3D4/21及3D4/21-p65^-/-细胞后PRV gB、gE蛋白的表达；滴度测定检测PRV-QXX感染3D4/21及3D4/21-p65^-/-细胞后子代病毒滴度。结果表明，sgRNA2和sgRNA3的基因编辑效率较高，对其进行克隆化培养进而获得敲除p65基因的稳定表达细胞系；CCK-8试剂盒检测细胞活力表明，p65基因敲除对细胞活力无影响；流式细胞仪检测表明，同一时间点PRV-GFP在3D4/21-p65^-/-中的增殖显著高于对照细胞；实时荧光定量PCR表明在3D4/21细胞中敲除p65基因促进了PRV gB、TK基因的mRNA表达水平，而抑制了IL-1β、IL-6基因的mRNA表达；Western blotting结果表明，在3D4/21细胞中敲除p65基因促进了PRV gB、gE蛋白的表达；滴度测定结果表明，同一时间点PRV-QXX在3D4/21-p65^-/-细胞中子代病毒的复制显著高于对照细胞。以上结果均表明，p65基因敲除可促进PRV在3D4/21细胞中复制。

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

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.