TRAF2和SNED1基因敲低对牛病毒性腹泻病毒复制的影响
Effect of TRAF2 and SNED1 Gene Knockdown on the Replication of Bovine viral diarrhea virus
刘芯怡 1权冉 1刘昱成 2陈俊贞 1倪慧莹 1付强 1史慧君1
作者信息
- 1. 新疆农业大学动物医学学院,乌鲁木齐 830052;新疆草食动物新药研究与创制重点实验室,乌鲁木齐 830052
- 2. 省部共建绵羊遗传改良与健康养殖国家重点实验室/新疆农垦科学院畜牧兽医研究所,石河子 832000
- 折叠
摘要
牛病毒性腹泻/粘膜病(bovine viral diarrhea/mucosal disease,BVD/MD)是由牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)引起的接触性传染病,会导致持续性感染,感染犊牛(Bos taurus)会不断向外界排出病毒.本课题组前期使用RNA-蛋白质相互作用检测(RNA-protein interaction detection,RaPID)技术,筛选出了与BVDV互作的肿瘤坏死因子受体相关因子2(tumor necrosis factor receptor associated factor 2,TRAF2)和Sushi、Nidogen和EGF样结构域1(Sushi,Nidogen and EGF like domains 1,SNED1)基因.为了确定TRAF2和SNED1基因对BVDV复制的影响,本研究利用小干扰RNA(small interfering RNA,siRNA)技术敲低牛肾细胞(madin-darby bovine kidney cells,MDBK)中TRAF2和SNED1基因,通过qPCR检测TRAF2和SNED1基因的敲低效率和BVDV感染TRAF2和SNED1敲低细胞的BVDV 5'UTR RNA水平变化;使用免疫荧光染色法检测BVDV复制的中间产物双链RNA(double stranded RNA,dsRNA)的积累变化;通过荧光倒置显微镜观察致细胞病变效应(cytopathic effect,CPE),使用Karber法计算子代病毒滴度变化.结果表明,TRAF2和SNED1的mRNA水平显著性降低(P＜0.05),表明成功构建了TRAF2以及SNED1敲低细胞(TRAF2 KD和SNED1 KD);BVDV感染对照组(NC)36 h开始出现明显的空斑、脱落、死亡等CPE现象,TRAF2 KD细胞和SNED1 KD细胞的CPE现象减弱;与对照组(NC)相比,在BVDV感染TRAF2 KD和SNED1 KD细胞后,BVDV 5'UTR RNA水平在24、36和48h显著下降(P＜0.05);通过免疫荧光染色检测BVDV复制的中间产物dsRNA形成和累积,在TRAF2 KD和SNED1 KD细胞中36和48h绿色荧光强度明显降低;BVDV感染TRAF2 KD和SNED1 KD细胞后48h子代病毒滴度极显著降低(P＜0.01).以上结果表明,敲低TRAF2和SNED1会抑制BVDV复制,本研究为揭示BVDV致病机制提供重要依据.
Abstract
Bovine viral diarrhea/mucosal disease(BVD/MD)is a contact infectious disease caused by Bovine viral diarrhea virus(BVDV),which will lead to persistent infection,and infected calves(Bos taurus)will continuously expel the virus to the outside.Our research group used RNA-protein interaction detection(RaPID)technology to screen tumor necrosis factor receptor associated factor 2,(TRAF2)and Sushi,Nidogen and EGF like domains 1(SNED1)genes.In order to determine the effects of TRAF2 and SNED1 genes on BVDV replication,in this study,the small interfering RNA(siRNA)technique was used to knock down the TRAF2 and SNED1 genes in madin-darby bovine kidney cells(MDBK),and the knock-down efficiency of TRAF2 and SNED1 genes and the changes of BVDV 5'UTR RNA level of BVDV infected cells were detected by qPCR.The accumulation of double stranded RNA(dsRNA),an intermediate product of BVDV replication,was detected by immunofluorescence staining.The cytopathic effect(CPE)was observed by fluorescence inverted microscope,and the virus titer of offspring was calculated by Karber method.The results showed that the mRNA levels of TRAF2 and SNED1 were significantly reduced(P＜0.05),which indicated that TRAF2 KD and SNED1 KD cells were successfully constructed.In BVDV infected control group(NC),CPE phenomena such as plaque,shedding and death began to appear at 36 h,and CPE phenomena of TRAF2 KD cells and SNED1 KD cells weakened.Compared with NC,after BVDV infected TRAF2 KD and SNED1 KD cells,the level of BVDV 5'UTR RNA decreased significantly at 24,36 and 48 h(P＜0.05).The formation and accumulation of dsRNA,an intermediate product of BVDV replication,were detected by immunofluorescence staining.The green fluorescence intensity in TRAF2 KD and SNED1 KD cells decreased significantly at 36 and 48 h.48 h after BVDV infected TRAF2 KD and SNED1 KD cells,the virus titer of the offspring decreased extremely significantly(P＜0.01).The above results showed that knocking down TRAF2 and SNED1 would inhibit BVDV replication,and this study provides an important basis for revealing the pathogenesis of BVDV.
关键词
牛病毒性腹泻病毒(BVDV)/小干扰RNA(siRNA)技术/肿瘤坏死因子受体相关因子2(TRAF2)/Sushi、Nidogen和EGF样结构域1(SNED1)Key words
Bovine viral diarrhea virus(BVDV)/Small interfering RNA(siRNA technology)/tumor necrosis factor receptor associated factor 2(TRAF2)/Sushi,Nidogen and EGF like domains 1(SNED1)引用本文复制引用
基金项目
国家自然科学基金(32060042)
国家自然科学基金(32160829)
新疆维吾尔自治区青年科技拔尖人才专项(2022TSYCCX0049)
新疆维吾尔自治区杰出青年基金(2022D01E15)
新疆维吾尔自治区研究生科研创新项目(XJ2022G131)
新疆农业大学大学生创新项目(dxscx2022183)
出版年
2024
国家科技期刊平台
NSTL
万方数据