干扰素基因刺激因子基因敲除对猪伪狂犬病病毒复制的影响  

Effect of Interferon Gene Stimulating Factor Gene Knockout on Replication of Porcine Pseudorabies Virus

作　　者：侯璐 王一 张爽 李国利 曾磊[1] 郭玉堃 翟云云 于朋伟 王棋 王春凤 杜永坤 万博[1] HOU Lu;WANG Yi;ZHANG Shuang;LI Guoli;ZENG Lei;GUO Yukun;ZHAI Yunyun;YU Pengwei;WANG Qi;WANG Chunfeng;DU Yongkun;WAN Bo(Key Laboratory of Animal Biochemistry and Nutrition of Ministry of Agriculture and Rural Affairs,Henan Agricultural University,Zhengzhou 450002,China)

机构地区：[1]河南农业大学农业部动物生化与营养重点开放实验室

出　　处：《畜牧兽医学报》2019年第11期2273-2282,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：转基因生物新品种培育重大专项(2016ZX08006001-006);优势特色学科建设经费(203/18xk0102);2019河南省重点研发与推广专项(192102110191);“十二五”农村领域国家科技计划课题(2015BAD12B02-3)

摘　　要：研究干扰素基因刺激因子(STING)基因敲除对猪伪狂犬病病毒(PRV)复制的影响。用CRISPR/Cas9技术敲除猪肺巨噬细胞系(3D4/21)的STING基因,用T7核酸酶检测靶基因敲除效率,用细胞计数试剂盒检测基因敲除细胞的活力,用表达绿色荧光蛋白(GFP)的重组病毒PRV-GFP感染基因敲除细胞,用流式细胞术检测感染细胞的荧光强度,用定量RT-PCR检测PRV的gB、TK基因表达及其感染细胞的IL-1β、IFN-β、ISG15基因转录,用Western blot检测PRV gE蛋白表达水平,用病毒滴定法测定子代病毒滴度。结果显示:设计的3个STING基因外显子2特异sgRNA均能切割3D4/21细胞的靶序列,其中sgRNA3的基因编辑效率最高;对sgRNA1介导STING基因敲除系进行克隆化,获得基因敲除细胞系3株,基因敲除对细胞活力无影响;亲本细胞培养的PRV-GFP感染阳性细胞占80.77%,STING基因敲除细胞培养的PRV-GFP感染阳性细胞占95.55%;STING基因敲除对PRV基因表达具有促进作用,亲本3D4/21细胞的病毒滴度为106.2 TCID50·0.1 mL^-1,STING基因敲除细胞的病毒滴度为108.3TCID50·0.1 mL^-1,病毒感染细胞的IL-1β、IFN-β和ISG15转录下调明显。STING基因敲除能促进PRV在3D4/21细胞中复制,可能与感染细胞的IL-β、IFN-β和ISG15表达抑制有关。This study was conducted to explore the effect of interferon gene stimulating factor(STING) gene knockout on replication of pseudorabies virus(PRV). The STING gene of porcine pulmonary macrophage cell line 3 D4/21 was knocked out by using CRISPR/Cas9 technique, the target gene knockout efficiency was detected by using T7 nuclease, and the activity of gene knockout cells was detected by using cell counting kit. The recombinant virus PRV-GFP expressing green fluorescent protein(GFP) was used to infect the knockout cells, and the fluorescence intensity of the infected cells was detected by flow cytometry. The expression of gB and TK genes in PRV and the transcription of IL-1β, IFN-β and ISG15 genes in infected cells were detected by quantitative RT-PCR, the expression level of PRV gE protein was detected by Western blot, and the viral titer of offspring was determined by viral titration. The results showed that all of three designed exon2 specific sgRNA of STING gene could cleave the target sequences of 3 D4/21 cells, and the gene editing efficiency of sgRNA3 was the highest. SgRNA1-mediated STING gene knockout lines were cloned and three gene knockout cell lines were obtained. Gene knockout had no effect on cell activity. PRV-GFP positive cells cultured in parental cells accounted for 80.77%, and PRV-GFP positive cells cultured in STING knockout cells accounted for 95.55%. STING gene knockout could promote the expression of PRV gene. The virus titer of parent 3 D4/21 cells was 106.2 TCID50·0.1 mL^-1, and the virus titer of STING knockout cells was 108.3 TCID50·0.1 mL^-1. The transcription of IL-1β, IFN-β and ISG15 in virus infected cells were significantly down-regulated. STING gene knockout can promote the replication of PRV in 3 D4/21 cells, which may be related to the inhibition of IL-1β, IFN-β and ISG15 expression in infected cells.

关 键 词：CRISPR/Cas9 干扰素基因刺激因子 猪肺巨噬细胞 伪狂犬病病毒 

分 类 号：S852.659.1[农业科学—基础兽医学]