血管内皮生长因子 (VEGF) 是一种特异性的促血管内皮细胞生长因子，与肿瘤侵袭、转移、预后和复发密切相关。本研究为表达筛选与 VEGF 具有亲和特异性的纳米抗体，首先利用 VEGF 免疫羊驼，提取外周血淋巴细胞总 RNA，构建噬菌体文库;并多次淘洗筛选到与 VEGF 有亲和力的克隆，筛选到 10 个与 VEGF 有亲和力的 VHH 基因片段。然后通过软件分析其亲水性、分子质量等理化性质，发现这 10 个 Nb_VEGF 蛋白均为亲水性蛋白，其分子质量在 20. 1 ～ 20. 7 kDa 之间。同时在大肠杆菌中重组表达这10 个抗体蛋白，经 Ni 金属螯合亲和层析纯化后的 Nb_VEGF 的蛋白纯度均大于 80%。最后采用酶联免疫吸附试验 (ELISA) 检测蛋白的抗原特异性和亲和力。结果表明: 有 3 个纳米抗体 Nb_VEGF-3、Nb_VEGF-7 和 Nb_VEGF-10 能与 VEGF 特异性结合，且 Nb_VEGF-3 的亲和力与阳性对照一致，其他纳米抗体均无 VEGF 抗性。本研究成功筛选并表达了 3 个具有VEGF 特异性的纳米抗体，且其中 Nb_VEGF-3 有较好的抗原亲和力，可能是一种抗 VEGF的候选药物。

ascular endothelial growth factor (VEGF) is a highly specific pro-vascular endothelial growth factor that closely related to tumor invasion，metastasis，prognosis and recurrence． This study is to screen and express the nanobodies with specificity and affinity for VEGF． Firstly，the llama was immunized with VEGF，and total RNA from peripheral blood lymphocytes was extracted to construct a phage library． The clones with affinity to VEGF were screened after repeated panning，and 10 VHH gene fragments with affinity to VEGF were obtained． Then their hy-drophilicity and molecular mass were analyzed by ExPASy，and it was found that 10 Nb_VEGF proteins were hydrophi-lic proteins with molecular masses between 20. 1kDa and 20. 7 kDa． At the same time，the 10 Nb_VEGF proteins were reconstructed in Escherichia coli，and the purity of the Nb VEGF proteins purified by Ni metal chelate affinity chroma-tography all exceeds 80%． Finally the specificity of the recombinant nanobodies was detected by enzyme linked im-munosorbent assay (ELISA)． The results suggests that three recombinant nanobodies，namely，Nb_VEGF-3，Nb_VEGF-7 and Nb_VEGF-10，can specifically bind with VEGF，the affinity of Nb_VEGF-3 is consistent with that of the positive control，and other nanobodies have no resistance． In this study，three nanobodies with specificity to VEGF were screened and expressed in E. coli successfully，and Nb_VEGF-3 shows good affinity with VEGF，so it could be a drug candidate against VEGF．