数字农科院2.0

鸡chNHE1精准基因编辑细胞系的构建及其抗ALV-J感染的研究

文献类型： 中文期刊

作者： 张学富;陈运通;范文瑞;张子博;于蒙蒙;王素艳;祁小乐;李留安;高玉龙

作者机构：

关键词： DF-1;chNHE1;ALV-J;基因编辑

期刊名称：畜牧兽医学报

ISSN： 0366-6964

年卷期： 2024 年

页码：

收录情况： 北大核心（2023版） ; ; CSCD（2023-2024年度） ; ; 科技核心（2024版） ; ; 农林核心（2020版）

摘要： 为构建ALV-J受体分子chNHE1精准基因编辑细胞系，本研究利用荧光标记的CRISPR/Cas9系统，在DF-1细胞中将chNHE1介导ALV-J进入宿主细胞的关键氨基酸V33进行突变、W38进行缺失，同时将编码第34-37位氨基酸的密码子同义替换。通过流式细胞分选获得48株单克隆细胞系，PCR及测序分析结果显示，其中有14株单克隆细胞系的chNHE1成功发生V33突变、W38缺失以及34-37位氨基酸的密码子同义替换，基因编辑效率为29%。为了验证chNHE1基因编辑DF-1细胞系的遗传稳定性及增殖水平，对传至第25代的细胞系进行测序分析，结果显示，chNHE1基因未发生回复性突变；进一步细胞计数分析结果显示，chNHE1基因编辑细胞系增殖水平未受到影响；为了评价chNHE1基因编辑细胞系抗ALV-J感染的能力，分别利用ALV-J荧光报告病毒（ALV-J-GFP）及ALV-J原型毒株（HPRS-103）对其进行病毒感染试验，荧光观察结果及流式细胞分析结果显示，chNHE1基因编辑细胞系可完全抵抗0.1 MOI ALV-J-GFP的感染；进一步间接免疫荧光试验、PCR扩增试验以及病毒滴度测定试验结果显示，chNHE1基因编辑细胞系可完全抵抗0.1 MOI HPRS-103毒株及0.1 MOI JL08CH3-1毒株的感染。本研究利用荧光标记的CRISPR/Cas9系统结合流式细胞分选，成功构建了chNHE1基因编辑细胞系，其可完全抵抗ALV-J的感染，且该细胞系遗传稳定性及增殖活性良好，为建立抗ALV-J感染的新技术提供了理论支持及基因编辑靶点。

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