西安交通大学学报（医学版）2017，Vol.38Issue(1)：7-12,6.DOI:10.7652/jdyxb201701002
靶向巨噬细胞膜蛋白Vsig4特异性纳米抗体的构建和筛选
Construction and screening of nanobody targeting macrophage membrane receptor Vsig4
摘要
Abstract
ABSTRACT:Objective To construct V-set and immunoglobulin domain containing 4 (Vsig4)nanobodies (Nbs) as specific macrophage probes so as to use them as molecular probes of macrophagocytes.Methods A nanobody phage library was generated by using peripheral blood lymphocytes isolated from an alpaca immunized with recombinant Vsig4 protein.After three rounds of selection against recombinant Vsig4.The Nbs were subjected to sequencing and genome alignment to obtain VHH sequence.Nbs were isolated and tested for Vsig4 specificity in an ELISA using recombinant Vsig4.The affinity capacity of Nbs was verified by the cell line stably expressing Vsig4. Results A nanobody phage library with an estimated 7.27 × 107 clones with 70% insertion was successfully constructed.Totally 1 3 6 Vsig4-positive clones were sequenced and aligned according to different CDR3 sequences. In summary,1 5 Vsig4 nanobodies were obtained and grouped into 3 different CDR3 epitopes.The affinity of representing nanobody and Vsig4 was analyzed via ELISA;Nb1 1 9 showed the highest affinity against both recombinant and native Vsig4.Conclusion We successfully constructed and screened Vsig4 specific nanobody number 1 1 9 with high affinity and specificity.It can help with macrophage detection and in vivo monitoring.关键词
Vsig4/纳米抗体/噬菌体展示文库/生物淘筛/亲和力鉴定Key words
Vsig4/phage display library/bio-banning/affinity determination分类
医药卫生引用本文复制引用
郑芳,罗思羽,韩燕,宁启兰,温玉荣..靶向巨噬细胞膜蛋白Vsig4特异性纳米抗体的构建和筛选[J].西安交通大学学报（医学版）,2017,38(1):7-12,6.基金项目
国家自然科学基金资助项目(No.81501527)、陕西省自然科学基础研究计划项目(No.XJJ2015048) Supported by the National Natural Science Foundation of China (No.81501527)and the Natural Science Basic Research Program of Shaanxi Province (No.XJJ2015048) （No.81501527）
