Preparation and identification of monoclonal antibodies against glycoprotein N glycoprotein of novel bunyavirus

ZHUGE Yaoyao; DONG Jing; TIAN Linshuang

doi:10.3969/j.issn.1671-8631.2025.4.sxlcyy202504011

Abstract：Objective:This study aimed to generate and characterize monoclonal antibodies against the N—terminal glycoprotein(Gn)of the severe fever with thrombocytopenia syndrome virus(SFTSV)strain JS2012-70,providing tools for further exploration of the virus's pathogenic mechanisms and the development of detection methods and thera-peutic strategies.Methods:The immunogenicity of the Gn protein was predicted using bioinformatics analysis.A recom-binant expression vector pET—28a(+)—Gn was constructed after removing the transmembrane domain.Gn protein was expressed using IPTG induction and purified through a nickel affinity chromatography column.The purified protein was used to immunize BALB/c female mice.Hybridoma and subcloning techniques were employed to screen hybridoma cell lines that stably secrete monoclonal antibodies against Gn.The specificity of the antibodies was validated by West-ern blot.Results:TheGn protein contains multiple antigenic epitopes,and its three—dimensional structure remained largely unchanged after the removal of the transmembrane domain.The recombinant Gn protein was successfully ex-pressed and purified at a concentration of 0.5 mg/mL.Four hybridoma cell lines(2C3,2F6,5E9,and 6C8)stably secre-ting monoclonal antibodies against Gn were identified.These antibodies exhibited ascitic fluid titers exceeding 1∶409600,were classified as IgG1/Kappa,and specifically recognized the Gn recombinant protein and SFTSV viral fluid in Western blot assays.Conclusion:This study successfully developed high—titer monoclonal antibodies against the Gn protein of SFTSV,laying the foundation for further research on the pathogenic mechanisms of SFTSV and the develop-ment of diagnostic and therapeutic strategies.