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目的 为深入探究Argonaute-2(AGO2)蛋白的生物学功能及其在抗病毒免疫中的作用，本研究通过构建Ago2基因敲除的小鼠神经母细胞瘤（Neuro 2a, N2a）细胞系（简称AKO细胞系）来验证其对弹状病毒科相关病毒复制的影响。方法通过设计靶向Ago2基因的sgRNA并将其连接至pMD18T-U6质粒中，随后将该质粒与pMJ920-Cas9-eGFP质粒共转染至小鼠神经母细胞瘤（Neuro 2a, N2a）中，培养36h后通过流式细胞筛选获得单克隆细胞，并利用分子生物学方法验证Ago2的有效敲除及敲除AGO2对细胞增殖与活力的影响。随后通过免疫印迹、RT-qPCR、病毒滴度测定验证敲除AGO2对狂犬病病毒（Rabies virus, RABV）和水疱口炎病毒（Vesicular stomatitis virus, VSV）复制的影响。结果 成功获得了一株AKO细胞系，该细胞系在细胞增殖与活力方面与野生型N2a细胞无显著差异。功能实验表明，敲除AGO2能够显著促进RABV核蛋白的蛋白、mRNA以及子代病毒滴度水平和VSV的子代病毒滴度水平。结论 本研究利用CRISPR/Cas9基因编辑技术和结合流式细胞分选相结合的方法，成功实现了在N2a细胞Ago2基因的快速、特异性敲除。通过功能实验表明，敲除AGO2能够促进RABV及VSV的复制。为阐明AGO2蛋白在细胞生理及抗病毒天然免疫中的功能提供了有效工具，也为基于RABV/VSV载体的重组病毒疫苗研发提供了实验依据。
Abstract：Objective To elucidate the biological functions of Argonaute-2(AGO2) and its role in antiviral innate immunity, this study aimed to generate an Ago2-knockout murine neuroblastoma(Neuro 2a, N2a) cell line(designated as the AKO cell line) and to investigate the effects of AGO2 deficiency on the replication of viruses belonging to the family Rhabdoviridae. Methods Single-guide RNAs(sgRNAs) targeting the Ago2 gene were designed and cloned into the pMD18T-U6 plasmid, which was subsequently co-transfected with the pMJ920-Cas9-eGFP plasmid into N2a cells. After 36 h of culture, monoclonal cell populations were isolated by flow cytometry sorting. Molecular biological assays were performed to confirm efficient Ago2 knockout and to assess the effects of AGO2 deficiency on cell proliferation and viability. Western blotting, RT-qPCR, and viral titration assays were then conducted to evaluate the impact of AGO2 knockout on the replication of rabies virus(RABV) and vesicular stomatitis virus(VSV). Results A stable Ago2-knockout N2a cell line(AKO) was successfully established. No significant differences in cell proliferation or viability were observed between AKO cells and wild-type N2a cells. Functional analyses revealed that AGO2 knockout markedly increased RABV nucleoprotein expression at both the protein and mRNA levels and significantly elevated progeny virus titers of RABV and VSV. Conclusion By combining CRISPR/Cas9-mediated genome editing with flow cytometry sorting, this study achieved rapid and specific knockout of Ago2 in N2a cells. Functional assays demonstrated that loss of AGO2 enhances the replication of RABV and VSV, indicating that AGO2 acts as a restriction factor against rhabdovirus replication. The AKO cell line represents a valuable experimental tool for investigating the roles of AGO2 in cellular physiology and innate antiviral immunity and provides an experimental basis for the development of recombinant viral vaccines based on RABV and VSV vectors.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250417
中图分类号:Q939.91
引用信息:
[1]李昊,李金斗,柳永赛,等.AGO2敲除N2a细胞系的构建及其对弹状病毒科病毒复制的影响[J].病毒学报,2026,42(02):583-591.DOI:10.13242/j.cnki.bingduxuebao.250417.
基金信息:
国家重点研发计划（项目号：2022YFD11800104）,题目：影响RABV感染与致病性的宿主限制因子的鉴定及作用机制研究; 吉林省青年科技人才培养项目（项目号：20250602022RC）,题目：宿主因子UPF1调控狂犬病病毒复制的分子机制研究~~
2025-12-24
2025
2026-02-24
2026
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2026-03-12
2026-03-12
2026-03-12