PK-15细胞的THBS3基因缺失抑制PRV复制
文献类型: 中文期刊
作者: 李志杰;;刘培欣;;杨 涛;;张 瑜;;宋海慧;;张 闯;;王 岳
作者机构:
关键词: THBS3;;PK-15细胞;;CRISPR/Cas9;;PRV
期刊名称:中国兽医学报
ISSN: 1005-4545
年卷期: 2023 年
页码:
收录情况: 北大核心(2020版) ; ; CSCD(2023-2024年度) ; ; 科技核心(2023版) ; ; 农林核心(2020版)
摘要: 为探索猪血小板反应蛋白(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,设计并合成两对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一株无THBS3蛋白表达细胞株(PK-THBS3-KO),通过测序发现外显子3有一个碱基的插入;感染实验证明THBS3基因缺失后抑制了PRV复制。本研究通过CRISPR/Cas9系统成功构建了PK-15的THBS3基因敲除的稳定细胞株,PRV感染实验显示基因缺失后抑制了病毒复制,为进一步研究THBS3在PRV感染中的机制奠定了基础。
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