军事医学2025，Vol.49Issue(6)：413-420,8.DOI:10.7644/j.issn.1674-9960.2025.06.003
基于mRNA免疫和单细胞测序技术筛选鉴定抗猴痘病毒E8L蛋白的单克隆抗体
Rapid selection and characterization of antibodies against monkeypox virus E8L based on mRNA immunization and single-cell sequencing
摘要
Abstract
Objective To screen and identify specific monoclonal antibodies targeting the surface envelope protein E8L of the monkeypox intracellular mature virus based on mRNA immunization and single-cell sequencing.Methods E8L-mRNA was synthesized in vitro and encapsulated into lipid nanoparticles(LNPs)for immunization to induce immune responses.The antibody level in blood serum was detected to find out when the mice had produced sufficient antibodies.B cells in the spleen of the mice were isolated by flow cytometry sorting,followed by library construction and sequencing.Based on the analysis of light and heavy chain sequences of the antibodies from the sequencing data,E8L candidate monoclonal antibodies were screened according to the abundance ranking and expressed.The affinity of these monoclonal antibodies was detected by enzyme-linked immunosorbent assay(ELISA).Candidate antibodies with high affinity for E8L were applied to immunochromatographic assays to detect the monkeypox virus(MPXV)E8L protein.Results Mice were immunized by lipid nanoparticles encapsulating E8L-mRNA.Approximately 1.5 million B cells were selected by flow cytometry.After single-cell sequencing,17 candidate monoclonal antibodies for E8L were identified and expressed.Two high affinity monoclonal antibodies were obtained for monkeypox virus E8L protein by ELISA.These two antibodies were used as the basis for immunochromatographic assays to detect E8L protein,with a sensitivity of 0.5 ng/mL.Conclusion Two monoclonal antibodies with high affinity for the MPXV E8L protein are obtained,which can be potentially used for detecting monkeypox virus.关键词
猴痘病毒/E8L抗原/单克隆抗体/mRNA免疫/单细胞测序Key words
monkeypox virus/antigen E8L/monoclonal antibody/mRNA immunization/single-cell sequencing分类
生物科学引用本文复制引用
缪伊琪,孙会胜,杨兴胜,张震,杨静..基于mRNA免疫和单细胞测序技术筛选鉴定抗猴痘病毒E8L蛋白的单克隆抗体[J].军事医学,2025,49(6):413-420,8.基金项目
国家重点研发计划(2021YFC2302405) （2021YFC2302405）
