中国兽医科学2025，Vol.55Issue(7)：853-861,9.DOI:10.16656/j.issn.1673-4696.2025.0117
RAB20基因敲除PK-15细胞系的构建及其对口蹄疫病毒复制的影响
Construction of RAB20-knockout PK-15 cell line and its impact on the replication of foot-and-mouth disease virus
摘要
Abstract
The aim of this study was to construct the RAB20 gene knockout porcine kidney cell line(PK-15)using CRISPR/Cas9 gene editing technology,to provide biological materials for the study of RAB20 protein function,and to initially explore the impact of RAB20 protein on the proliferation of foot-and-mouth disease virus(FMDV).First of all,2 pairs of sgRNA were designed and synthesized according to the RAB20 gene sequence,which were constructed into the pX459 plasmid respectively.The recombinant plasmids were transfected into PK-15 cells,screened with purinomycin,and subcloned by limited dilution method.Then,monoclonal cells sequencing and Western-blot were used to identify the knockout of the RAB20 gene,and CCK-8 was used to determine the effect of RAB20 knockout on cell vitality.Then,RAB20 knockout cells and wild-type cells were infected with FMDV,and Western-blot,real-time fluorescence quantitative PCR,indirect immunofluorescence test and viral titre determination were used to compare the replication differences of FMDV between knockout cell lines and wild-type cells.The results showed that the RAB20 gene knockout PK-15 cell line has been successfully constructed,and compared with wild cells,the replication level of FMDV in RAB20 knockout cells has been significantly improved,indicating that RAB20 has an inhibitory effect on the replication of FMDV.The results provide cell models and data support for further research on the function of RAB20 and its mechanism of regulating virus replication.关键词
CRISPR/Cas9/RAB20基因/细胞系/口蹄疫病毒Key words
CRISPR/Cas9/RAB20 gene/cell line/foot-and-mouth disease virus分类
农业科技引用本文复制引用
蔡建涛,张伟,陈治彤,刘泓屹,吕岳芹,曹伟军,冷非凡,杨帆,郑海学..RAB20基因敲除PK-15细胞系的构建及其对口蹄疫病毒复制的影响[J].中国兽医科学,2025,55(7):853-861,9.基金项目
国家自然科学基金项目(32072831,32102639) （32072831,32102639）
国家重点研发计划项目(2021YFD1800300) （2021YFD1800300）
甘肃省杰出青年基金项目(21JR7RA026) （21JR7RA026）
兰州大学中央高校基本科研业务费专项(lzujbky-2022-ey20) （lzujbky-2022-ey20）
甘肃省科技重大专项(22ZD6NA001) （22ZD6NA001）
