中国兽医科学2026，Vol.56Issue(1)：33-39,7.DOI:10.16656/j.issn.1673-4696.2026.0001
猪源TRAF3基因编辑细胞系的构建及缺失表型研究
Construction and deletion phenotype study of porcine TRAF3 gene edited cell line
摘要
Abstract
This study utilized CRISPR/Cas9 technology to construct a PK-15 pig kidney cell model with TRAF3 gene knockout,which provides experimental materials for elucidating the regulatory role of TRAF3 in the replication of foot-and-mouth disease virus(FMDV).Two highly efficient and specific sin-gle guide RNAs(sgRNAs)were designed and screened against the coding region sequence of the porcine TRAF3 gene and were respectively cloned into the CRISPR/Cas9 vector pX459.After the recombinant plas-mids were introduced into PK-15 cells by liposome-mediated transfection technology,a monoclonal cell line was successfully obtained by combining the strategy of puromycin gradient screening and limiting dilution method.The constructed cell line was verified by DNA sequencing combined with Western-blot.The results showed that there were specific base deletion mutations in the TRAF3 gene,and the expres-sion level of the corresponding protein was undetectable,thus confirming the successful construction of the TRAF3 gene-knockout cell model.To explore the regulatory effect of this gene deletion on FMDV replication,multi-dimensional detections such as Western-blot,real-time quantitative PCR(RT-qPCR),and 50％tissue culture infectious dose(TCID50)were carried out.The results showed that the absence of TRAF3 protein could enhance the replication ability of FMDV.These findings not only provide a novel tool cell line for clarifying the biological function of TRAF3 but also lay an experimental foundation for subsequent research on the molecular mechanism of this gene on virus-host interactions.关键词
CRISPR/Cas9/TNF受体关联蛋白3/细胞系/口蹄疫病毒Key words
CRISPR/Cas9/TNF receptor associated factor 3/cell lines/foot-and-mouth disease virus分类
农业科技引用本文复制引用
邵文华,杨帆,曹伟军,陈创伟,王伟,郑海学,张伟,朱紫祥,赵宗胜..猪源TRAF3基因编辑细胞系的构建及缺失表型研究[J].中国兽医科学,2026,56(1):33-39,7.基金项目
国家自然科学基金项目(32473064) （32473064）
国家重点研发计划项目(2021YFD1800302) （2021YFD1800302）
兰州大学中央高校基本科研业务费专项资金(lzujbky-2022-ey20) （lzujbky-2022-ey20）
国家生猪产业技术体系项目(CARS-35) （CARS-35）
国家生猪技术创新中心项目(NCTIP-XD/C03) （NCTIP-XD/C03）
甘肃省自然科学基金项目(23JRRA547) （23JRRA547）
