| 李 洁,徐元宏.基于活细胞显微技术的登革病毒膜融合实验方法的建立及初步应用[J].安徽医药,2015,19(11):2127-2130. |
| 基于活细胞显微技术的登革病毒膜融合实验方法的建立及初步应用 |
| Development and preliminary application of experimental method for dengue virus membrane fusion based on live cell microscopy |
| 投稿时间：2015-06-19 |
| DOI： |
| 中文关键词: 登革病毒 中和抗体 膜融合 |
| 英文关键词: dengue virus neutralizing antibody membrane fusion |
| 基金项目:安徽省自然科学基金青年科学基金项目(No 1508085QH176) |
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| 中文摘要: |
| 目的 建立一种基于活细胞显微技术的膜融合实验方法,分析抗体在登革病毒(Dengue virus,DENV)包膜糖蛋白E(envelope glycoprotein)介导的细胞膜融合中的作用。方法 DENV感染C6/36蚊子细胞,48 h后酸化细胞内涵体环境以诱导病毒与细胞发生膜融合,同时加入不同稀释浓度的中和抗体；观察抗体在病毒—宿主细胞膜融合中所起到的作用；借助Incell Analyzer 2000高内涵细胞成像分析系统拍摄图片并用软件进行定量分析。结果 在宿主细胞内涵体环境酸性化(pH值6.0)后的100 min时能清晰地观察到登革病毒与C6/36细胞发生膜融合；中和抗体4G2及9F12均能抑制膜融合(IC50分别为0.02、0.03 g·L-1),最终中和登革病毒感染。结论 运用此方法,能测定登革病毒抗体的膜融合抑制能力,从而了解其中和作用机制。 |
| 英文摘要: |
| Objective To develop a membrane fusion assay based on live cell microscopy technique and to investigate the role of antibodies in dengue virus-cell membrane fusion mediated by envelope glycoprotein.Methods C6/36 mosquito cells were infected with DENV for 48 h, and then the acidification of endosomal environment was performed to induce virus-cell membrane fusion. Meanwhile, serial diluted neutralizing antibodies were added into cells and the role of antibodies in virus-cell membrane fusion was observed. By using the Incell analyzer 2000 high content cell imaging analysis system and its software, pictures of live cells were taken and the results were analyzed quantitatively.Results In the acidic environment of host cell endosome (pH 6.0), the membrane fusion of dengue virus and C6/36 mosquito cells can be clearly observed at 100 minutes after pH drop. Neutralizing antibody 4G2 and 9F12 can inhibit membrane fusion (IC50 of 0.02 and 0.03 g·L-1, respectively), and thus block the viral infection.Conclusions By using this new method, the ability of antibody against DENV to inhibit the membrane fusion can be determined and the neutralization mechanism would be further understood. |
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