SARS-CoV-2非感染性细胞-细胞融合模型的构建  

Construction of SARS-CoV-2 cell-cell fusion model

作　　者：单静 杨曼清 尚方建 刘奇 SHAN Jing;YANG Manqing;SHANG Fangjian;LIU Qi(Integrated Lab of Pathology Biology,Collegeof Basic Medical,Dali University,Dali 671000,Yunnan,China)

机构地区：[1]大理大学基础医学院病原生物学综合实验室,云南大理671000

出　　处：《中国病原生物学杂志》2023年第6期625-629,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.81703573);大理大学创新团队项目(No.ZKLX2019105)。

摘　　要：目的构建一种基于SARS-CoV-2S蛋白介导的非感染性、可视化细胞-细胞融合模型,用于检测SARS-CoV-2进入抑制剂的抑制活性。方法构建可同时表达SARS-CoV-2S蛋白和绿色荧光蛋白EGFP的重组质粒pAAVEGFP-SARS2-S,在293T细胞瞬时表达(293T/EGFP/SARS-2),将其与靶细胞Huh-7共同培养,模拟病毒侵染靶细胞,分别在2、8、12、24、48h于荧光显微镜下观察并记录细胞不同时间的融合情况,同时在该模型上评价SARS-CoV-2进入抑制剂EK1的活性。结果荧光显微镜观察293T/EGFP/SARS-2和Huh-7细胞混合后的细胞融合情况:4h后发生融合,24h后可形成大面积融合。SARS-CoV-2进入抑制剂EK1能抑制细胞融合,且呈剂量依赖,其IC50为(287±1.78)nmol/L。结论成功构建无感染性、成本低廉的SARS-CoV-2进入抑制剂药物检测筛选融合模型,可对天然小分子化合物进行筛选,为冠状病毒药物的研发提供了一种检测途径。Objective To construct a non-infectious and visual cell-cell fusion model mediated by SARS-CoV-2 S protein to detect the inhibitory activity of SARS-CoV-2 entry inhibitors.Methods The recombinant plasmid pAAV-EGFP-SARS2 was constructed to express both SARS-CoV-2 S protein and green fluorescent protein EGFP.The recombinant plasmid PaAV-EGFP-SARS2 was transient expressed in 293T cells(293T/EGFP/SARS-2)and co-cultured with target cell Huh-7 to simulate virus infection of target cells.Fluorescence microscopy was used to observe and record the fusion of cells at different times at 2,8,12,24 and 48 h.Meanwhile,the activity of SARS-CoV-2 entry inhibitor EK1 was evaluated on this model.Results Fluorescence microscope observation of cell fusion after mixing 293T/EGFP/SARS-2 and Huh-7 cells:fusion occurred 4 hours later,and large-scale fusion could be formed 24 hours later.The entry inhibitor EK1 of SARS-CoV-2 can inhibit cell fusion in a dose-dependent manner,with an IC50 of(287±1.78)nmol/L.Conclusion Successfully constructed a non-infectious and low-cost SARS-CoV-2 inhibitor drug detection and screening fusion model,which can screen natural small molecule compounds and provide a detection pathway for the development of coronavirus.

关 键 词：SARS-CoV-2 细胞-细胞融合 进入抑制剂 

分 类 号：R373.1[医药卫生—病原生物学]