囊泡运输蛋白SEC22B敲低对牛病毒性腹泻病毒复制的影响研究  

Effect of Vesicle Transport Protein SEC22B Knockdown on the Replication of Bovine viral diarrhea virus

作　　者：张成远 袁圆圆 郭妍婷 杨莉 冉多良[1] 付强[1] 史慧君[1] ZHANG Cheng-Yuan;YUAN Yuan-Yuan;GUO Yan-Ting;YANG Li;RAN Duo-Liang;FU Qiang;SHI Hui-Jun(College of Animal Medicine,Xinjiang Agricultural University,Urumqi 830052,China)

机构地区：[1]新疆农业大学动物医学学院,乌鲁木齐830052

出　　处：《农业生物技术学报》2023年第4期784-790,共7页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31902271;32060042);自治区国际合作项目(2020E01006);自治区百名博士引进计划。

摘　　要：牛病毒性腹泻病的感染情况日趋严重,给养殖业造成了严重的损失,目前我国缺乏有效的治疗手段,导致感染模式复杂化,其致病机制和病毒-宿主之间相互作用研究也尚不清楚。为了探究囊泡运输蛋白(vesicle transport protein homolog,SEC22B)敲低对牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)复制的影响,根据GenBank数据库中SEC22B基因序列,使用Benchling平台设计向导RNA(small guide RNA,sgRNA),克隆至LentiCRISPR v2载体后进行慢病毒包装,使用CRISPR/Cas9基因编辑技术敲低胎牛肾细胞(madin-darby bovine kidney cells,MDBK)中的SEC22B基因表达,使用Western blot鉴定SEC22B基因敲低(knockdown,KD)情况;BVDV感染SEC22B KD细胞不同时间后,分别使用qPCR和免疫荧光染色检测BVDV 5’UTR mRNA水平变化和双链RNA(double-stranded RNA,dsRNA)量,使用Reed-Muench法测定BVDV感染不同时间后子代病毒滴度变化。Western blot检测发现,与对照Scramble细胞相比,SEC22B KD细胞中SEC22B蛋白表达量显著降低;与BVDV感染Scramble细胞相比,BVDV感染SEC22B KD细胞后5’UTR mRNA水平和dsRNA量均极显著降低(P<0.01),同时,子代病毒滴度极显著下降(P<0.01)。结果表明,敲低SEC22B基因后显著抑制BVDV复制,本研究为BVDV防控新方法的建立提供理论依据。The infection of bovine viral diarrhea is becoming more and more serious,which has caused serious losses to the breeding industry.At present,the lack of effective treatment leads to the complicated infection pattern in China,and the research on the pathogenesis and virus-host interaction is still unclear.To investigate the effect of vesicle transport protein(SEC22B)deletion on Bovine viral diarrhea virus(BVDV)replication,according to the SEC22B gene sequence in GenBank database,small guide RNA(sg RNA)was designed using Benchling platform and cloned into LentiCRISPR V2 vector for lentivirus packaging.The SEC22B gene in madin-darby bovine kidney cells(MDBK)was knocked down(KD)by CRISPR/Cas9 gene editing technique,and identified by Western blot.After BVDV infection with SEC22B KD cells at different times,the changes of 5’UTR mRNA level and accumulation of double-stranded RNA(dsRNA)were detected by qPCR and immunofluorescence staining,respectively.Reed-Muench method was used to measure the titer change of progeny virus after BVDV infection at different time.The results showed that compared with scramble cells,the protein expression level of SEC22B in SEC22B KD cells was significantly decreased by Western blot.Compared with scramble cells infected with BVDV,the level of 5’UTR mRNA and ds RNA of SEC22B KD cells were extremely significantly decreased(P<0.01)after BVDV infection,and the titer of progeny virus was extremely significantly decreased(P<0.01).The results showed that knockdown SEC22B gene significantly inhibited the replication of BVDV.This study provides a target for the establishment of new methods for the prevention and control of BVDV.

关 键 词：囊泡运输蛋白(SEC22B) 牛病毒性腹泻病毒(BVDV) CRISPR/Cas9基因编辑技术 基因敲低 复制 

分 类 号：S852.65[农业科学—基础兽医学]