HCoV-229E细胞-细胞融合模型构建  

Construction of a cell-cell fusion model mediated by HCoV-229E

作　　者：杨曼清 单静 尚方建 刘奇 YANG Manqing;SHAN Jing;SHANG Fangjian;LIU Qi(School of Basic Medical Sciences,Dali University,Dali 671000,China)

机构地区：[1]大理大学基础医学院,云南大理671000

出　　处：《生物技术》2023年第6期702-707,726,共7页Biotechnology

基　　金：国家自然科学基金项目(81703573,81660337);大理大学创新团队项目(ZKLX2019105)。

摘　　要：[目的]构建一种HCoV-229E侵染靶细胞的可视化细胞-细胞融合模型。[方法]构建真核表达质粒pAAV-IRSE-GFP-229E S。转染293T细胞后,该细胞可以模拟病毒与靶细胞Huh-7发生融合。在此基础上使用冠状病毒广谱抑制剂EK1检测抑制效果。[结果]成功构建pAAV-GFP-229E S重组质粒。转染293T后可表达绿色荧光蛋白GFP,与Huh-7混合培养2 h后可在荧光显微镜下观察到融合现象(形态增大、荧光强度减弱、呈现不规则状);48 h后形成更大面积的融合。EK1以浓度依赖的方式抑制HCoV-229E S介导的细胞-细胞融合,IC50为(3265.948±719.116)nmol/L。[结论]成功构建了HCoV-229E侵染靶细胞的细胞-细胞融合模型,用于体外靶向HCoV-229E S蛋白的药物筛选。[Objective]To construct a visual cell-cell fusion model of HCoV-229E infected target cells.[Method]The eukaryotic expression plasmid pAAV-IRSE-GFP-229E S was constructed.After transfection of 293T cells,the cells were able to simulate the fusion of the virus with the target cell Huh-7.On this basis,coronavirus broad-spectrum inhibitor EK1 was used to detect the inhibitory effect.[Result]pAAV-GFP-229E S recombinant plasmid was constructed successfully.Green fluorescent protein GFP was expressed after transfection with 293T,and the fusion phenomenon(increased morphology,decreased fluorescence intensity and irregular appearance)was observed under fluorescence microscope after 2 hours mixed culture with Huh-7.After 48 hours,a larger area of fusion was formed.EK1 inhibited HCoV-229E S-mediated cell-cell fusion in a concentration-dependent manner with an IC_(50)of(3265.948±719.116)nmol/L.[Conclusion]The cell-cell fusion model of HCoV-229E infected target cells was successfully constructed for in vitro drug screening targeting HCoV-229E S protein.

关 键 词：中医药 冠状病毒 HCoV-229E 细胞-细胞融合 靶向S蛋白 进入抑制剂 抗病毒药物 药物筛选 

分 类 号：R373.1[医药卫生—病原生物学]