摘要
试验旨在研究敲除干扰素调节因子3 (interferon regulatory factor 3,IRF3)对伪狂犬病病毒(pseudorabies virus,PRV)复制的影响。使用慢病毒介导的CRISPR/Cas9基因编辑技术建立了IRF3基因敲除PK15细胞系。通过构建重组质粒pIRF3-sgRNA,转染至HEK293T/17细胞,收获慢病毒并感染PK15细胞,经嘌呤霉素筛选获得多克隆细胞系,T7酶切鉴定后通过有限稀释法获得PK15-IRF3^(-/-)单克隆稳定细胞系。为验证敲除IRF3基因稳定细胞系是否构建成功,采用实时荧光定量PCR、Western blotting方法检测PRV相关基因及蛋白的表达,应用荧光显微镜和流式细胞术观察病毒复制情况并进行病毒滴度检测。结果显示,PK15-IRF3^(-/-)细胞系感染PRV-GFP后PK15-IRF3^(-/-)细胞荧光强度明显强于PK15细胞。感染PRV野毒(PRV-QXX)后PK15-IRF3^(-/-)病毒滴度明显强于PK15细胞,PRV的gE蛋白水平明显高于PK15细胞。在mRNA水平检测两种细胞中PRV TK基因的变化也得到相同的结果。进一步研究表明,在感染PRV-QXX后,PK15细胞中IFN-βmRNA会随着时间增加显著增高(P<0.05),但PK15-IRF3^(-/-)细胞中IFN-βmRNA则无明显变化。上述结果表明,敲除IRF3基因后显著促进PRV的增殖,IRF3在病毒的复制中发挥着重要作用,为伪狂犬病的防控提供了新的方法和策略。
The aim of the study was to investigate the effect of knockout of interferon regulatory factor 3(IRF3) on the replication of pseudorabies virus(PRV).The IRF3 knockout PK15 cell line was established using lentiviral-mediated CRISPR/Cas9 genome editing technology.The recombinant plasmid pIRF3-sgRNA was constructed and transfected into HEK293 T/17 cells.Lentivirus was obtained and infected with PK15 cells.The polyclonal cell line was obtained by puromycin screening.After T7 digestion,PK15-IRF3-/- monoclonal stable cell line was obtained by limiting dilution method.To verify whether the stable cell line of IRF3 gene was successfully constructed,Real-time PCR and Western blotting were used to detect the expression of PRV-related genes and proteins.Fluorescence microscopy and flow cytometry were used to observe the virus replication.The results showed that the fluorescence intensity of PK15-IRF3-/- cells was significantly stronger than that of PK15 cells after infection of PRV-GFP in PK15-IRF3-/- cell line.The PK15-IRF3-/- virus titer was significantly stronger than that of PK15 cells after infection with PRV-QXX,and the gE protein level of PRV was significantly higher than that of PK15 cells.The same results were obtained by detecting changes of PRV TK gene in the two cells at the mRNA level.Further studies showed that IFN-β mRNA in PK15 cells increased significantly with time after infection with PRV-QXX(P<0.05),but there was no significant change in IFN-β mRNA in PK15-IRF3-/- cells.The above results indicated that knocking out IRF3 significantly promoted the proliferation of PRV,and IRF3 played an important role in virus replication,providing new methods and strategies for the prevention and control of pseudorabies.
作者
张爽
樊爽爽
常雯茹
丁光绪
郭瑞珍
段利芳
杜永坤
褚贝贝
杨国宇
王江
ZHANG Shuang;FAN Shuangshuang;CHANG Wenru;DING Guangxu;GUO Ruizhen;DUAN Lifang;DU Yongkun;CHU Beibei;YANG Guoyu;WANG Jiang(Key Laboratory of Animal Biochemistry and Nutrition,Ministry of Agriculture and Rural Affiars,Henan Agricultural University,Zhengzhou 450002,China)
出处
《中国畜牧兽医》
CAS
北大核心
2019年第5期1253-1262,共10页
China Animal Husbandry & Veterinary Medicine
基金
转基因生物新品种培育重大专项(2016ZX08006001-006)
国家自然科学基金(31502031)
霍英东教育基金会高等院校青年教师基金(151033)
优势特色学科建设经费(203/18xk0102)
