人源性抗SARS-CoV-2单链抗体文库的构建及广谱中和抗体的筛选与鉴定  

Construction of a human anti-SARS-CoV-2 scFv library and identification of broad-spectrum neutralizing antibodies

作　　者：尹慧敏 吕海 迟莹[2] 刘静娴[2] 焦永军[2] 卫平民 YIN Huimin;LYU Hai;CHI Ying;LIU Jingxian;JIAO Yongjun;WEI Pingmin(Key Laboratory of Environmental Medicine Engineering,Ministry of Education,School of Public Health,Southeast University;Key Laboratory of Enteric Pathogenic Microorganisms,Jiangsu Provincial Center for Disease Control and Prevention,Nanjing 210009,China)

机构地区：[1]东南大学公共卫生学院环境医学工程教育部重点实验室 [2]江苏省疾病预防控制中心卫生与健康委员会肠道病原微生物重点实验室,江苏南京210009

出　　处：《细胞与分子免疫学杂志》2025年第2期154-160,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：南京市重大科技专项(2021-11005);江苏省基础研究计划自然科学基金面上项目(BK20221413)。

摘　　要：目的构建人源性抗严重急性呼吸综合征冠状病毒2(SARS-CoV-2)单链可变片段(scFv)抗体文库,并筛选具有广谱中和活性的抗体,为诊断和治疗制剂的开发提供候选分子。方法从新型冠状病毒感染康复者外周血中分离单个核淋巴细胞(PBMC),提取总RNA,逆转录成cDNA,以此为模板构建人源性抗SARS-CoV-2 scFv抗体文库;利用噬菌体展示技术筛选对SARS-CoV-2 S蛋白具有特异性的scFv抗体。通过序列分析和人源IgG抗体表达,合成IgG全抗体,并评估其对SARS-CoV-2的结合能力及中和活性。结果成功构建了库容量为1.56×10^(7)CFU的SARS-CoV-2人源性scFv抗体文库,筛选出两种特异性scFv抗体;并将scFv抗体表达成IgG形式的全抗体(IgG-A10和IgG-G6),其中IgG-A10对SARS-CoV-2原始株(WT)和奥密克戎亚型变异毒株XBB(XBB)毒株均具有较好中和活性,对奥密克戎BA.2.86变异株的第二代亚分支JN.1(JN.1)毒株中和效果一般。结论本研究从康复者外周血中成功构建了人源性抗SARS-CoV-2 scFv抗体文库,并筛选表达出具有中和活性的抗SARS-CoV-2 IgG抗体,为SARS-CoV-2感染的预防、诊断和治疗奠定了基础。Objective To construct a library of human-derived anti-severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)single-chain variable fragments(scFv)and screen for broad-spectrum neutralizing antibodies to identify candidate molecules for the development of diagnostic and therapeutic agents.Methods Peripheral blood mononuclear cells(PBMCs)were isolated from the peripheral blood of patients who had recovered from novel coronavirus infection.Total RNA was extracted from these PBMCs and reverse transcribed into cDNA,which was used as a template for constructing a human anti-SARS-CoV-2 scFv library.Phage display technology was used to screen for scFv antibodies specific to the SARS-CoV-2 S protein.Full-length IgG antibodies were synthesized through sequence analysis and human IgG expression,and their binding capacity and neutralizing activity against SARS-CoV-2 were evaluated.Results A human-derived scFv antibody library against SARS-CoV-2 with a capacity of 1.56×10^(7)CFU was successfully constructed.Two specific scFv antibodies were screened from this library and expressed as full-length IgG antibodies(IgG-A10 and IgG-G6).IgG-A10 exhibited strong neutralizing activity against both the original SARS-CoV-2 strain(WT)and the XBB subvariant of the Omicron variant.However,the neutralizing activity of this antibody against the JN.1 sub lineage of the Omicron BA.2.86 variant was moderate.Conclusion This study has successfully constructed a human anti-SARS-CoV-2 scFv antibody library from the peripheral blood of recovered patients,and screened and expressed anti-SARS-CoV-2 IgG antibodies with neutralizing activity,laying a foundation for the prevention,diagnosis,and treatment of SARS-CoV-2 infection.

关 键 词：SARS-CoV-2 单链可变片段(scFv) 噬菌体展示技术 中和抗体 

分 类 号：R392[医药卫生—免疫学] R714.253[医药卫生—基础医学] S852.43[农业科学—基础兽医学]