PK-15细胞的THBS3基因缺失抑制PRV复制

THBS3 deficiency in PK-15 cell inhibits PRV replication

为探索猪血小板反应蛋白3(thrombospondin 3,THBS3)在PRV复制中的功能,利用CRISPR/Cas9系统构建THBS3基因缺失的PK-15稳定细胞株。首先,根据THBS3基因序列和CRISPR/Cas9靶点设计原则设计并合成2对sgRNA序列,退火后连接到Lenti-CRISPRv2慢病毒表达载体;与慢病毒包装质粒共转染人胚肾细胞(HEK293T)获得重组慢病毒,收集细胞上清并离心去细胞碎片后转导PK-15细胞,进行嘌呤霉素筛选获得THBS3敲除的PK-15细胞株;将重组病毒rPRV-GFP接种至野生型和缺失型细胞评价病毒复制差异。结果显示,表达sgRNA的重组质粒构建成功;将收获的慢病毒转导细胞后通过筛选获得1株无THBS3蛋白表达细胞株(PK-THBS3-KO),通过测序发现外显子3有1个碱基的插入;感染试验显示,THBS3基因缺失后抑制了PRV复制。结果表明,通过CRISPR/Cas9系统成功构建了PK-15的THBS3基因敲除的稳定细胞株,PRV感染试验显示基因缺失后抑制了病毒复制。

In order to explore the function of thrombospondin 3(THBS3) in pseudorabies virus(PRV)replication,CRISPR/Cas9 system was used to construct PK-15 cell strains with THBS3 deficiency.First,a sgRNA targeting the THBS3 gene was designed according to the principle for CRISPR/Cas9 sgRNA.After annealing and producing double strands sgRNA,the recombinant plasmid expressing sgRNA was constructed by cloning to Lenti-CRISPRv2.The recombinant lentivirus was produced by co-transfecting sgRNA expression plasmid and packaging plasmids into human embryonic kidney cell(HEK293T).Recombinant lentiviruses were collected andtransduced into PK-15 cells,and PK-15 cell strains with THBS3 deficiency were obtained by puromycin screening.rPRV-GFP was inoculated into wild-type and mutation cells to evaluate the virus replication efficiency.The results showed that the recombinant plasmid expressing sgRNA was constructed successfully,and was transduced into PK-15 cells.After screening,a stable PK-15 cell strain without THBS3 expression was abtained.Infection experiments showed that PRV replication was inhibited in the THBS3 gene knockout PK-15 cells.In this study,a stable PK-15 cell strain with THBS3 gene knockout was successfully constructed by CRISPR/Cas9 system.PRV infection experiment showed that virus replication was inhibited in this cell strain,which laid a foundation for further study on the mechanism of how THBS3 affects PRV infection.