VEGF纳米抗体的筛选、表达及特异性检测

Screening,Expression and Specificity Detection of Anti-VEGF Nanobodies

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。

Vascular 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 hydrophilicity and molecular mass were analyzed by ExPASy,and it was found that 10 Nb VEGF proteins were hydrophi-lic proteins with molecular masses between 20.1 kDa 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 chromatography all exceeds 80%.Finally the specificity of the recombinant nanobodies was detected by enzyme linked immunosorbent 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.