农业生物技术学报2024，Vol.32Issue(9)：2150-2158,9.DOI:10.3969/j.issn.1674-7968.2024.09.017
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
摘要
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)分类
农业科技引用本文复制引用
刘芯怡,权冉,刘昱成,陈俊贞,倪慧莹,付强,史慧君..TRAF2和SNED1基因敲低对牛病毒性腹泻病毒复制的影响[J].农业生物技术学报,2024,32(9):2150-2158,9.基金项目
国家自然科学基金(32060042 （）
32160829) （）
新疆维吾尔自治区青年科技拔尖人才专项(2022TSYCCX0049) （2022TSYCCX0049）
新疆维吾尔自治区杰出青年基金(2022D01E15) （2022D01E15）
新疆维吾尔自治区研究生科研创新项目(XJ2022G131) （XJ2022G131）
新疆农业大学大学生创新项目(dxscx2022183) （dxscx2022183）
