猪源TRAF3基因编辑细胞系的构建及缺失表型研究OA

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.