| 赵雅坤,魏销玥,孟凡亮,刘文韬,范佳铭,龙丽瑾,王婉婷,陈建伶,张建中,何利华,刘立雍,赵瑞,孙迪,袁雪珍,闫笑梅.大容量天然人源Fab噬菌体展示抗体库的构建及鉴定[J].中华流行病学杂志,2025,46(2):288-295 |
| 大容量天然人源Fab噬菌体展示抗体库的构建及鉴定 |
| Construction and identification of a sizeable naive human Fab phage display antibody library |
| 收稿日期:2024-08-01 出版日期:2025-02-14 |
| DOI：10.3760/cma.j.cn112338-20240801-00468 |
| 中文关键词: 噬菌体展示抗体库 Fab抗体 天然抗体库 |
| 英文关键词: Phage display antibody library Antigen-binding fragment Naive antibody library |
| 基金项目:首都卫生发展科研专项（2024-2G-4363）；国家自然科学基金面上项目（81873959）；财政专项基金（102393240020020000003） |
| 作者 | 单位 | E-mail | | 赵雅坤 | 中国医科大学公共卫生学院卫生统计学教研室, 沈阳 110122
传染病溯源预警与智能决策全国重点实验室, 中国疾病预防控制中心传染病预防控制所, 北京 102206 | | | 魏销玥 | 传染病溯源预警与智能决策全国重点实验室, 中国疾病预防控制中心传染病预防控制所, 北京 102206 | | | 孟凡亮 | 传染病溯源预警与智能决策全国重点实验室, 中国疾病预防控制中心传染病预防控制所, 北京 102206 | | | 刘文韬 | 传染病溯源预警与智能决策全国重点实验室, 中国疾病预防控制中心传染病预防控制所, 北京 102206 | | | 范佳铭 | 传染病溯源预警与智能决策全国重点实验室, 中国疾病预防控制中心传染病预防控制所, 北京 102206 | | | 龙丽瑾 | 中国医科大学公共卫生学院卫生统计学教研室, 沈阳 110122
传染病溯源预警与智能决策全国重点实验室, 中国疾病预防控制中心传染病预防控制所, 北京 102206 | | | 王婉婷 | 传染病溯源预警与智能决策全国重点实验室, 中国疾病预防控制中心传染病预防控制所, 北京 102206 | | | 陈建伶 | 传染病溯源预警与智能决策全国重点实验室, 中国疾病预防控制中心传染病预防控制所, 北京 102206 | | | 张建中 | 传染病溯源预警与智能决策全国重点实验室, 中国疾病预防控制中心传染病预防控制所, 北京 102206 | | | 何利华 | 传染病溯源预警与智能决策全国重点实验室, 中国疾病预防控制中心传染病预防控制所, 北京 102206 | | | 刘立雍 | 传染病溯源预警与智能决策全国重点实验室, 中国疾病预防控制中心传染病预防控制所, 北京 102206 | | | 赵瑞 | 北京知禾新创生物技术有限公司, 北京 102299 | | | 孙迪 | 北京知禾新创生物技术有限公司, 北京 102299 | | | 袁雪珍 | 北京知禾新创生物技术有限公司, 北京 102299 | | | 闫笑梅 | 传染病溯源预警与智能决策全国重点实验室, 中国疾病预防控制中心传染病预防控制所, 北京 102206 | yanxiaomei@icdc.cn |
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| 中文摘要: |
| 目的 构建大容量天然人源Fab噬菌体展示抗体库，用于体外筛选高亲和力的特异性抗体。方法 收集126名健康人的外周血，分离单个核细胞，提取RNA，反转录合成cDNA，以cDNA为模板，通过PCR分别扩增IgG、IgM的VH以及抗体Vκ、Cκ和Vλ、Cλ基因，以第一次PCR产物为模板进行二次PCR。通过重叠PCR将Vκ 与 Cκ 及 Vλ 与 Cλ 连接得到轻链，最后通过重叠PCR得到轻链+VH片段。将酶切的抗体基因与含有重链CH1 编码基因的噬菌体载体pNC3连接，电转化大肠埃希菌TG1感受态细胞。随机挑选100个阳性克隆进行菌落PCR鉴定及测序，并利用IMGT数据库和MAFFT序列比对软件分析抗体基因多态性。以重组表达的金黄色葡萄球菌α溶血素对抗体库进行富集筛选。随机挑选单克隆进行噬菌体ELISA检测，并对阳性克隆（抗原A450∶空白对照A450≥2.1）进行测序。结果 成功构建2个库容量分别为1.25×1011的κ链和1.54×1011的λ链的大容量天然人源Fab噬菌体展示抗体库，κ链和λ链噬菌体展示抗体库噬菌体滴度分别为6.04×1013CFU/ml和3.50×1013CFU/ml。κ链和λ链噬菌体展示抗体库转化阳性插入率分别为96％（96/100）和100％（100/100）。序列分析发现，测序获得的抗体序列均为独特序列。骨架区氨基酸序列相对保守，互补决定区（CDR）氨基酸序列长度不等，氨基酸多样性大，尤其是CDR3。经过IMGT数据库分析发现，序列有广泛的可变-多样性-连接片段家族分布。经过6轮富集筛选，获得了针对金黄色葡萄球菌α溶血素的特异性噬菌体抗体富集，对142个单克隆进行测序，结果显示共有8条独特型Fab抗体序列。结论 本研究成功构建了大容量天然人源Fab噬菌体展示抗体库，抗体库多样性良好，可用于血清流行病学相关的抗体筛选。 |
| 英文摘要: |
| Objective To construct a sizeable naive human Fab phage display antibody library to screen high-affinity specific antibodies in vitro. Methods Total RNA was extracted from peripheral blood mononuclear cells (PBMCs) of 126 healthy individuals, subsequently reverse-transcribed into cDNA, and used as a template. PCR amplification was performed to obtain the VH from IgG, IgM and light chain κ, λ, separately, with the initial PCR products serving as templates for a second round of PCR. Overlap extension PCR was employed to generate fragments of the κ and λ light chains. These fragments were ligated with the phage vector pNC3, which harbors the variable region 1 of the heavy chain, to construct a recombinant phage plasmid. This plasmid was then electroporated into competent Escherichia Coli TG1 cells to establish a naive human Fab phage display antibody library. One hundred clones were randomly selected for identification and sequencing, and antibody gene polymorphisms were analyzed using the IMGT database and MAFFT software. Recombinant α-hemolysin from Staphylococcus aureus was utilized to screen Fab antibody fragments through biopanning of the antibody library, followed by random selection of phage ELISA-identified clones. The positive clones (antigen A450∶blank control A450≥2.1) were sequenced. Results Two large naive Fab phage display antibody libraries were successfully constructed, in which the capacity of κ and λ chain antibody libraries were 1.25×1011and 1.54×1011, respectively. The titers for two antibody libraries were 6.04×1013 CFU/ml and 3.50×1013 CFU/ml. The positive transformation insertion rates for κ and λ chain antibody libraries were 96% (96/100) and 100% (100/100), respectively. Sequence analysis revealed that all antibody sequences were unique. The amino acid sequences in the skeletal region were relatively conserved. In contrast, significant variations in the length of the complementarity determining region (CDR) were found, and the diversity of amino acid sequence of the complementary determining region was high, especially the CDR3. Analysis using the IMGT database indicated that the sequences exhibited a broad distribution across variable-diversity-joining gene families. After six rounds of panning, specific phage antibodies enrichment targeting α-hemolysin were achieved. A total of 142 monoclonal antibodies were sequenced, yielding 8 distinct Fab antibody sequences. Conclusion This study successfully constructed two naive human Fab phage display antibody libraries with large capacity and good diversity, which can be used for screening human antibodies for serum epidemiology. |
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