数字农科院2.0

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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