关键词: 纳米抗体, 血管内皮生长因子, 噬菌体文库, 筛选

Abstract: 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．

Key words: nanobody, vascular endothelial growth factor, phage library, screening