摘要
目的研制和筛选识别艰难梭菌(Clostridium difficile)谷氨酸脱氢酶(glutamate dehydrogenase,CD-GDH)线性B细胞抗原表位的驼源单域抗体,并将其应用于Western blot和ELISA等检测方法。方法以重组表达并纯化的CD-GDH作为诱饵,从噬菌体展示驼源天然纳米抗体库中筛选阳性噬菌体克隆。经Western blot验证后测定抗体基因序列,然后密码子优化合成抗体基因序列并克隆至pET28a载体中构建表达载体。经诱导表达和纯化后应用于Western blot和ELISA检测CD-GDH蛋白。结果共获得6个驼源单域抗体阳性克隆,其中克隆GA4被选择在大肠埃希菌中重组表达并纯化。GA4与CD-GDH结合的亲和力良好,K_(d)值为3 nmol/L,应用于Western blot和ELISA都能特异性地识别重组表达和内源性的CD-GDH蛋白。结论成功制备并获得了1株识别CD-GDH蛋白中线性B细胞抗原表位的驼源单域抗体,为检测CD-GDH蛋白提供了有效工具。
Objective To develop a camelid single-domain antibody(SdAb)recognizing linear B-cell epitopes in glutamate dehydrogenase of Clostridium difficile(CD-GDH),and to apply it in Western blot and ELISA.Methods Purified recombinant CD-GDH was used as bait to screen phage-displayed camelid SdAb library and obtain positive clones.Then those clones were confirmed by Western blot,and their variable domain of heavy chain of heavy chain antibody(VHH)nucleotide sequence were determined.The VHH sequence was synthesized after codon optimization and cloned into the expression vector pET28a.The SdAb was then expressed and purified,and its ability to detect CD-GDH protein in multiple assays was further explored.Results Six positive clones were obtained,among which clone GA4 was chosen for recombinant expression in Escherichia coli and further purification.The purified GA4 binded well with CD-GDH with a K_(d) value of 3 nmol/L.In Western blot and ELISA,GA4 was proven to be able to selectively detect both recombinant and endogenous CD-GDH.Conclusions A camelid SdAb targeting a linear B-cell epitope in CD-GDH is successfully developed,which provides a very useful tool for detecting CD-GDH.
作者
翟华倩
李哲舟
蔡梦婷
张凯
沈立军
罗永能
金大智
胡珲
Zhai Huaqian;Li Zhezhou;Cai Mengting;Zhang Kai;Shen Lijun;Luo Yongneng;Jin Dazhi;Hu Hui(School of Laboratory Medicine,Hangzhou Medical College,Hangzhou 310053,China;Department of Hepatobiliary and Pancreatic Surgery,the First Affiliated Hospital,Zhejiang University School of Medicine,Hangzhou 310009,China)
出处
《中华微生物学和免疫学杂志》
北大核心
2025年第8期629-635,共7页
Chinese Journal of Microbiology and Immunology
基金
国家级大学生创新训练计划项目(202413023031)
国家自然科学基金(82100696)。
关键词
艰难梭菌
谷氨酸脱氢酶
抗原表位
驼源
单域抗体
Clostridium difficile
Glutamate dehydrogenase
Epitope
Camelid
Single-domain antibody
