Screening and identification of human Zn T8-specific single-chain variable fragment (scFv) from type

• 作 者: Qian Wu Xiaodong Wang Yong Gu Xiao Zhang Yao Qin Heng Chen Xinyu Xu Tao Yang Mei Zhang
• 机 构: Department of Endocrinology, The First Affiliated Hospital of Nanjing Medical University,Key Laborat
• 出 处: Science China(Life Sciences)
• 发表日期: 2016年07期
• 关键词: phage library scFv Screening affinity immunotherapy islet dimer purification tra

Zinc transporter 8(ZnT8) is a major autoantigen and a predictive marker in type 1 diabetes(T1D). To investigate ZnT8-specific antibodies, a phage display library from T1 D was constructed and single-chain antibodies against ZnT 8 were screened and identified. Human T1 D single-chain variable fragment(sc Fv) phage display library consists of approximately 1í10~8 clones. After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated the highest affinity to ZnT8, were expressed in Escherichia coli Top10F’ and then purified by affinity chromatography. C27 and C22 specifically bound ZnT8 N/C fusion protein and ZnT8 C terminal dimer with one Arg325 Trp mutation. The specificity to human islet cells of these sc Fvs were further confirmed by immunohistochemistry. In conclusion, we have successfully constructed a T1 D phage display antibody library and identified two ZnT8-specific sc Fv clones, C27 and C22. These ZnT8-specific sc Fvs are potential agents in immunodiagnostic and immunotherapy of T1 D.

Zinc transporter 8 (ZnT8) is a major autoantigen and a predictive marker in type 1 diabetes (T1D). To investigate ZnT8-specific antibodies, a phage display library from T1 D was constructed and single-chain antibodies against ZnT8 were screened and identified After four rounds of bio-panning, seven unique clones were positive by phage ELISA. Among them, C27 and C22, which demonstrated that the phage display library consists of approximately 1 10 10 ~ 8 clones. the highest affinity to ZnT8, were expressed in Escherichia coli Top10F ’and then purified by affinity chromatography. C27 and C22 specifically bound ZnT8 N / C fusion protein and ZnT8 C terminal dimer with one Arg325 Trp mutation. The specificity to human islet cells of these sc Fvs were further confirmed by immunohistochemistry. In conclusion, we have successfully constructed a T1 D phage display antibody library and identified two ZnT8-specific sc Fv clones, C27 and C22. These ZnT8-specific sc Fvs are potential agents in immunodiagnostic and immunotherapy of T1D.