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：目的 构建V-set and immunoglobulin domain containing 4(Vsig4)特异性纳米抗体，以期作为巨噬细胞的分子探针。 方法 用Vsig4重组蛋白对羊驼进行免疫，分离血液中的淋巴细胞，利用噬菌体展示技术，构建噬菌体展示文库，经过连续3次生物淘筛获得与Vsig4蛋白结合的噬菌体，经测序和基因比对所得VHH序列，用ELISA法筛选出抗Vsig4的高亲和力纳米抗体，并用Vsig4稳定表达细胞系验证纳米抗体的结合能力。结果 成功构建了插入率为70%、库容量为7.27×107的噬菌体表达文库，经过克隆筛选获得136个Vsig4阳性单克隆，经测序获得15个不同的VHH基因，将这些基因克隆至原核表达体系，表达和纯化后获得了高纯度的Vsig4纳米抗体，其中Nb119的亲和力最高，并且可以与Vsig4稳定表达细胞系结合。结论 成功构建并筛选了特异性、高亲和力的Vsig4纳米抗体，以期用于检测巨噬细胞表面Vsig4的表达和构建特异性分子探针。
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 136 Vsig4-positive clones were sequenced and aligned according to different CDR3 sequences. In summary, 15 Vsig4 nanobodies were obtained and grouped into 3 different CDR3 epitopes. The affinity of representing nanobody and Vsig4 was analyzed via ELISA; Nb119 showed the highest affinity against both recombinant and native Vsig4. Conclusion? ?We successfully constructed and screened Vsig4 specific nanobody number 119 with high affinity and specificity. It can help with macrophage detection and in vivo monitoring