干扰STAT1基因表达对狂犬病病毒感染的影响  

Effects of interfering with STAT1 gene expression on rabies virus infection

作　　者：栗朵朵 蔡宗伶 张宏燕 李晓宁[1,2,3] 罗廷荣 LI Duo-duo;CAI Zong-ling;ZHANG Hong-yan;LI Xiao-ning;LUO Ting-rong(College of Animal Sciences and Technology,Guangxi University,Nanning,Guangxi 530004,China;State Key Laboratory of Subtropical Agricultural Biological Resources Protection and Utilization,Nanning,Guangxi 530004,China;Guangxi Engineering Research Center of Veterinary Biologics/Guangxi Key Laboratory of Livestock Breeding and Disease Control and Prevention,Nanning,Guangxi 530004,China)

机构地区：[1]广西大学动物科学技术学院,广西南宁530004 [2]亚热带农业生物资源保护利用国家重点实验室,广西南宁530004 [3]广西兽用生物制品工程研究中心/广西畜禽繁育与疾病防控重点实验室,广西南宁530004

出　　处：《南方农业学报》2024年第12期3718-3727,共10页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(32070161);国家重点研发计划项目(2022YFD1800101)。

摘　　要：【目的】探讨信号转导和转录激活因子1(STAT1)在狂犬病病毒(RABV)感染过程中的作用机制,为后续STAT1功能研究提供理论依据。【方法】以RABV感染小鼠小脑星形胶质细胞(C8-D1A),于接毒后12、24和48 h分别采用实时荧光定量PCR和蛋白免疫印迹(Western blotting)检测病毒基因及宿主细胞STAT1和细胞因子的表达变化;针对STAT1基因靶序列设计合成不同的siRNA序列,通过脂质体法瞬时转染C8-D1A细胞,筛选出干扰效率较高的STAT1-siRNA序列;然后以筛选出的STAT1-siRNA序列转染C8-D1A细胞,转染24 h后接种0.1 MOI的RABV,分别从转录水平和蛋白水平检测干扰STAT1基因表达对RABV复制及细胞因子表达的影响。【结果】RABV感染C8-D1A细胞后能引起STAT1蛋白的表达大幅度上升;无论是弱毒株rRC-HL还是标准强毒株CVS-11感染,都能引起C8-D1A细胞内IFN-α、IFN-β、TNF-α和IL-6等细胞因子基因上调表达。经STAT1-siRNA干扰效果验证,发现在最高浓度200 nmol/L下作用24 h后,STAT1-1616的干扰效率为81%、STAT1-2271的干扰效率为82%,干扰效果达极显著水平(P<0.01);作用48 h后,STAT1-1616的干扰效率为83%,STAT1-2271的干扰效率为75%,干扰效果达显著水平(P<0.05),故选择这2对STAT1-siRNA进行后续试验。干扰STAT1基因表达后,无论是接种弱毒株rRC-HL还是接种标准强毒株CVS-11,其N和P基因的表达均呈上调趋势,病毒N、P蛋白在接毒后24和36 h均呈上调表达趋势,说明干扰STAT1基因表达有利于上调病毒蛋白合成,而促进RABV复制;但干扰STAT1基因表达对RABV感染引起宿主细胞产生IFN-α、IFN-β、TNF-α和IL-6等细胞因子无显著影响(P>0.05)。【结论】合成的2对STAT1-siRNA序列(STAT1-1616和STAT1-2271)对C8-D1A细胞的STAT1基因有显著干扰效果。干扰STAT1基因表达有利于上调病毒蛋白合成,而促进RABV复制。【Objective】To explore the mechanism of signal transducer and activator of transcription 1(STAT1)in the process of rabies virus(RABV)infection,which could provide theoretical basis for subsequent research on STAT1 function.【Method】After murine-derived astrocytes(C8-D1A)infected with RABV,the expression changes of virus genes,host cell STAT1 and cytokines were detected by real-time fluorescence quantitative PCR and western blotting at 12,24 and 48 h post infection.Different siRNA sequences were designed and synthesized according to the STAT1 gene target sequence,and the liposome was instantaneously transfected into C8-D1A cells.The STAT1-siRNA sequences with the higher interference efficiency were screened for further experiments.The selected STAT1-siRNA sequences were transfected into C8-D1A cells,and then the cells were inoculated with 0.1 MOI RABV at 24 h post infection,the effects of interference with STAT1 gene expression on RABV replication and cytokine expression were detected at transcription level and protein level respectively.【Result】RABV infection on C8-D1A cells could cause large increase in the expression of STAT1 protein.Both the attenuated strain rRC-HL and the standard virulent strain CVS-11 infection could cause upregulation of cytokines genes including IFN-α,IFN-β,TNF-αand IL-6 in C8-D1A cells.Through the verification of the interference effect of STAT1-siRNA,it was found that after 24 h of action at the highest concentration of 200 nmol/L,the interference efficiency of STAT1-1616 was 81%,and the interference efficiency of STAT1-2271 was 82%,with extremely significant level of interference effect(P<0.01).After 48 h of action,the interference efficiency of STAT1-1616 was 83%,and the interference efficiency of STAT1-2271 was 75%,with significant level of interference effect(P<0.05).Therefore,these 2 pairs of STAT1-siRNA were selected for subsequent experiments.After interfering with the expression of STAT1 gene,both attenuated strain rRC-HL and the standard virulent strain CVS-11 sho

关 键 词：狂犬病病毒(RABV) 信号转导和转录激活因子1基因(STAT1) RNA干扰 干扰效率 细胞因子 

分 类 号：S852.659.5[农业科学—基础兽医学]