基于VBIM技术筛选RSV复制相关宿主基因
Screening Host Genes Associated with RSV Replication by VBIM
朱英霞 1杜作苑 1吴星奇 1黄鹤1
作者信息
- 1. 中国医学科学院病原生物学研究所 北京 102629
- 折叠
摘要
目的:利用基于慢病毒的可验证的插入突变(validation-based insertional mutagenesis,VBIM)技术筛选与呼吸道合胞病毒(respiratory syncytial virus,RSV)复制相关的宿主因子.方法:包装VBIM慢病毒转导HEp-2细胞形成突变细胞池,以RSV为筛选压力获得RSV抵抗性细胞克隆,提取细胞基因组DNA;通过反向PCR,鉴定抵抗性克隆VBIM慢病毒插入位点.利用siRNA敲减相应基因表达,通过RT-qPCR、western blot等分析RSV复制情况.结果:筛选获得6个RSV抵抗性细胞克隆,鉴定出AKR1C3、DDX18、GFI1B、TAPBP、MEF2A、SEMA3C、KCNIP4、GRID2等8个潜在调控病毒复制的宿主基因.经初步验证,基因MEF2A敲减后RSV RNA水平下降21％,蛋白水平下降60％,RSV滴度下降68％;SEMA3C敲减后,RSV RNA水平下降23％,蛋白水平下降28％,RSV滴度下降40％.结论:成功利用VBIM技术筛选出影响RSV复制的宿主基因,可能是参与RSV复制的宿主基因,为研究病毒与宿主相互作用及RSV防治提供理论基础.
Abstract
Objective:To employ the validation-based insertional mutagenesis(VBIM)technology in screening host factors asso-ciated with respiratory syncytial virus(RSV).Methods:HEp-2 cells were transduced with VBIM lentiviruses to generate a mutant cell pool.RSV-resistant cell clones were obtained after RSV infection.After extracting genomic DNA from these cells,we used reverse PCR to identify inserted sites of the VBIM vector in the resistant clones.Corresponding gene siRNAs were synthesized and used to suppress the gene expression.The replication of RSV was then analyzed using RT-qPCR and western blotting,the viral titers were determined as well.Results:Six RSV-resistant cell clones were obtained and eight potential host genes regulating RSV replication including AKR1C3,DDX18,GFI1B,TAPBP,MEF2A,SEMA3C,KCNIP4,GRID2 were identified.After preliminary verification,RSV RNA level,protein level and viral titer decreased by 21％,60％and 68％respectively after MEF2A knockdown.After SEMA3C knockdown,RSV RNA level decreased by 23％,protein level decreased by 28％,and viral titer decreased by 40％.Conclusions:By utilizing the VBIM technol-ogy,we successfully obtained host factors affecting RSV replication,providing a theoretical basis for the study of virus-host interaction as well as RSV prevention and treatment.
关键词
呼吸道合胞病毒/VBIM筛选/宿主因子Key words
Respiratory syncytial virus/VBIM screening/Host factors引用本文复制引用
出版年
2024
NETL
NSTL
万方数据