中国兽医科学2026，Vol.56Issue(4)：443-449,7.DOI:10.16656/j.issn.1673-4696.2026.0064
CRISPR/Cas9介导的ALKBH5基因敲除细胞系的建立及其应用
Establishment and application of a CRISPR/Cas9-mediated ALKBH5 knockout cell line
摘要
Abstract
This study aimed to clarify the role and mechanism of the ALKBH5 gene in foot-and-mouth disease virus(FMDV)replication through its m6A demethylase activity.A BHK-21 cell line with ALKBH5 knockout was successfully established using CRISPR/Cas9 technology,and the knockout efficiency was confirmed by Western-blot.The CCK-8 assay indicated no significant change in cell viability after knockout.Following FMDV infection of wild-type and knockout cells,it was found that ALKBH5 knockout significantly enhanced the expression of the viral 2C protein,viral RNA transcription levels,and viral titers,suggesting that ALKBH5 deletion promoted FMDV replication.Further enzymatic activity rescue experiments demonstrated that complementation with wild-type ALKBH5 suppressed viral replication,whereas the key catalytic site mutant(R131A)lost this inhibitory activity,confirming that the inhibitory effect of ALKBH5 on FMDV replication depended on its demethylase activity.This study reveals that ALKBH5 functions as a host restriction factor that negatively regulates FMDV replication through m6A demethylation,providing a theoretical basis for further elucidating the mechanisms of viral infection and developing novel antiviral drugs.关键词
ALKBH5/口蹄疫病毒/细胞系Key words
ALKBH5/foot-and-mouth disease virus/cell line分类
农业科技引用本文复制引用
任青峰,万博,李伟伟,裴丹诗,王一卓,何路,朱兆宇,李熙忠,郑海学,范许许..CRISPR/Cas9介导的ALKBH5基因敲除细胞系的建立及其应用[J].中国兽医科学,2026,56(4):443-449,7.基金项目
国家自然科学基金青年基金项目(32302857,32202781) （32302857,32202781）
国家重点研发计划项目(2021YFD1800300) （2021YFD1800300）
国家自然科学基金重点项目(32330107) （32330107）
中国博士后科学基金项目(2023M743830) （2023M743830）
国家生猪产业技术体系项目(CARS-35,CARS-39-13) （CARS-35,CARS-39-13）
国家生猪技术创新中心先导科技项目(NCTIP-XD/C03) （NCTIP-XD/C03）
中国农业科学院创新计划项目(CAAS-CSLPDCP-2023002,CAAS-ASTIP-2023-LVRI) （CAAS-CSLPDCP-2023002,CAAS-ASTIP-2023-LVRI）
"十四五"广东省农业科技创新十大主攻方向"揭榜挂帅"项目(2024KJ14) （2024KJ14）
中国农业科学院兰州兽医研究所基本科研业务项目(1610312021009) （1610312021009）
