摘要
目的通过单细胞测序筛选柯萨奇A组16型病毒(Coxsackievirus A16,CVA16)中和性单克隆抗体,并对其功能进行验证。方法以CVA16衣壳蛋白VP1作为抗原蛋白,流式细胞术分选VP1特异识别小鼠B细胞,对其进行B细胞抗原受体(B-cell receptor,BCR)组库单细胞测序;基于测序数据分析IgG抗体轻重链序列,根据丰度排序筛选CVA16候选单克隆抗体并进行表达;利用酶联免疫吸附实验(enzyme-linked immuno sorbent assay,ELISA)、微量中和实验等方法,对CVA16单克隆抗体进行体内外功能验证。结果成功表达CVA16-VP1-His重组蛋白,共免疫14只ICR小鼠,经流式分选出约50000个存活VP1特异B细胞,单细胞测序构建BCR库,筛选并表达19株CVA16候选单克隆抗体。经验证单抗7-2、7-4、7-8、8-8及8-9具有中和作用,半数抑制率(half maximal inhibitory concentration,IC50)分别为9.09、12.00、5.56、9.68、9.67μg/mL。单抗7-8在体外细胞水平上具有较强的中和作用,在体内乳鼠攻毒保护实验中也表现出良好的中和效果。广谱性中和实验表明,单抗7-1具有针对EVA71病毒的中和能力,单抗7-5显示出针对CVA6病毒的中和能力。结论本研究成功建立了1种基于单细胞测序技术筛选CVA16单克隆抗体的方法,验证抗体7-8为1株具备较强中和活性的单克隆抗体。
Objective:The aim of this study was to screen neutralizing monoclonal antibodies against Coxsackievirus A group 16(CVA16)through single-cell sequencing and to validate their functions.Methods:CVA16 capsid protein VP1 was used as the antigenic protein,and specific B cells recognizing VP1 were then sorted by flow cytometry and sequenced by BCR library single-cell sequencing.Based on the analysis of the IgG antibody light and heavy chain sequences from the sequencing data,CVA16 candidate monoclonal antibodies were screened according to the abundance ranking and expressed.The in vivo and in vitro functions of the identified CVA16 monoclonal antibodies were verified by enzyme-linked immunosorbent assay(ELISA)and micro-neutralization assays.Results:The CVA16-VP1-His recombinant protein was successfully expressed.Approximately 50000,12.00 viable VP1-specific B cells were sorted out by flow cytometry,and the BCR library was constructed and sequenced.19 candidate monoclonal antibodies against CVA16 were screened.Monoclonal antibodies 7-2,7-4,7-8,8-8,and 8-9 have neutralizing activity,with IC50 values of 9.09,12.00,5.56,9.68,and 9.67μg/mL,respectively.Monoclonal antibody 7-8 showed strong neutralization at the cellular level in vitro and also showed good neutralization in the in vivo challenge protection assay in Suckling mice.In addition,monoclonal antibody 7-1 had neutralizing ability against EVA71 virus,and 7-5 showed neutralizing ability against CVA6 virus.Conclusion:This study successfully established a pipeline for screening CVA16 monoclonal antibodies based on single-cell sequencing and found that antibody 7-8 had a strong neutralizing activity.
作者
李静
马传敏
全传松
李玉明
张振杰
史卫峰
LI Jing;MA Chuanmin;QUAN Chuansong;LI Yuming;ZHANG Zhenjie;SHI Weifeng(School of Clinical and Basic Medicine Sciences,Shandong First Medical University,Jinan 250117,China;Jinan Center for Disease Control and Prevention,Jinan 250021,China;School of Public Health,Shandong First Medical University,Jinan 250117,China;Ruijin Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 200025,China;Shanghai Institute of Virology,Shanghai Jiao Tong University School of Medicine,Shanghai 200025,China)
出处
《山东第一医科大学(山东省医学科学院)学报》
2025年第1期1-8,共8页
Journal of Shandong First Medical University & Shandong Academy of Medical Sciences
基金
国家自然科学基金(32470157)
山东省自然科学基金(ZR2021MC001)
泰山学者工程专项(tsqn202306261)
山东第一医科大学临床-基础联合创新团队项目(202407)。
