宿主蛋白Rab11a对牛病毒性腹泻病毒复制的影响
Effect of Host Protein Rab11a on Replication of Bovine Viral Diarrhea Virus
权冉 1葛丽娟 1陈俊贞 1袁圆圆 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
- 折叠
摘要
为了探索宿主蛋白Rab11a在感染牛病毒性腹泻病毒(BVDV)后,对细胞增殖的影响.通过CRISPR/Cas9基因编辑技术构建Rab11a基因稳定敲除的MDBK细胞株,设计靶向Rab11a基因的向导RNA表达载体,采用Western blot检测Rab11a基因敲除后的蛋白表达情况;使用实时荧光定量qPCR、免疫荧光染色试验、Reed-Muench法等技术检测BVDV感染Rab11a KO细胞后病毒RNA水平、双链RNA(dsRNA)积累水平、病毒滴度及致细胞病变(CPE)情况等.Western blot检测结果显示,成功构建出Rab11a基因敲除的细胞Rab11a KO;Rab11a基因敲除显著抑制BVDV 5'UTR mRNA和dsRNA水平的积累,感染36 h后,Scramble细胞明显有大量细胞病变,出现皱缩、碎裂、形成空泡后脱落等现象,Rab11a KO细胞的CPE现象明显低于Scramble细胞;在BVDV感染细胞12 h后,与Scramble细胞相比,Rab11a KO细胞的病毒滴度显著性降低,结果证明了敲除MDBK细胞的Rab11a基因对BVDV在胞内复制起到抑制作用.本研究结果为了解宿主蛋白Rab11a与BVDV的相互作用,以及Rab11a对病毒的复制增殖的作用提供了新的科学依据.
Abstract
To investigate the effect of host protein Rab11a on the proliferation of Bovine viral diarrhea virus(BVDV),the MDBK cell line with stable Rab11a gene knockout was constructed by CRISPR/Cas9 gene editing technology and the guide RNA expression vector targeting Rab11a gene was designed in the present study.The protein expression of Rab11a gene knockout was examined by Western blot.The levels of viral RNA and double strand RNA,virus titer and CPE were tested in qPCR and immunofluorescence assay.The results showed that Rab11a KO cells with the gene knockout were constructed as demonstrated in Western blot and Rab11a knockdown significantly inhibited the accumulation of BVDV 5'UTR mRNA and dsRNA levels.A large number of cytopathic changes were observed in the Scramble cells at 36 h post BVDV infection,such as shrinkage,fragmentation,vacuolation and fall off,etc.The CPE development was significantly lower in Rab11a KO cells than Scramble cells.In addition,the BVDV titer in Rab11a KO cells was significantly lower than that of Scramble cells at 12 h post infection.In conclusion,knockout of Rab11a gene in MDBK cells inhibited the intracellular replication of BVDV.The interaction between host protein Rab11a and BVDV was more understood from the discovery in this study.
关键词
Rab11a/牛病毒性腹泻病毒/CRISPR/Cas9/复制Key words
Rab11a/Bovine viral diarrhea virus/CRISPR/Cas9/replication引用本文复制引用
基金项目
国家自然科学基金(31902271)
国家自然科学基金(32060042)
自治区国际合作项目(2020E01006)
自治区创新团队项目(2020D14005)
自治区百名博士引进计划()
出版年
2024
国家科技期刊平台
NSTL
万方数据