College of Animal Science and Veterinary Medicine, Shanxi Agriculture University, Taigu, Jinzhong, 030801, Shanxi, People's Republic of China.
Mol Biol Rep. 2020 Mar;47(3):2137-2147. doi: 10.1007/s11033-020-05313-w. Epub 2020 Feb 20.
The aim of the study was to produce a single-domain antibody (nanobody) specific for endothelin receptor type B (EDNRB) which has high expression in melanoma. Cultured human melanoma cells were used as antigens to immunize alpacas. After antibody generation was verified in alpaca serum, total RNA was extracted from alpaca lymphocytes and the target VH fragment was amplified by two-step PCR, cloned in the pCANTAB5E phagemid vector, and used to transform Escherichia coli TG1 cells to obtain a phage-display nanobody library, which was enriched by panning. The results indicated successful construction of a phage-display anti-human melanoma A375 nanobodies library with a size of 1.2 × 10/ml and insertion rate of 80%. After screening, eight positive clones of anti-EDNRB nanobodies were used to infect E. coli HB2151 for production of soluble nanobodies, which were identified by ELISA. Finally, we obtained a high-affinity anti-EDNRB nanobody, which consisted of 119 amino acids (molecular weight: 12.97 kDa) with 22 amino acids in CDR3 and had good affinity in vitro. The results suggest that the nanobody may be potentially used for the treatment of human melanoma.
本研究旨在制备一种针对在黑色素瘤中高表达的内皮素受体 B（EDNRB）的单域抗体（纳米抗体）。用人黑色素瘤细胞作为抗原对羊驼进行免疫。在羊驼血清中验证抗体产生后，从羊驼淋巴细胞中提取总 RNA，通过两步 PCR 扩增目标 VH 片段，克隆到 pCANTAB5E 噬菌粒载体中，并转化大肠杆菌 TG1 细胞，获得噬菌体展示纳米抗体文库，然后通过淘选进行富集。结果表明成功构建了一个大小为 1.2×10/ml、插入率为 80%的噬菌体展示抗人黑色素瘤 A375 纳米抗体文库。经过筛选，得到了 8 株抗 EDNRB 纳米抗体的阳性克隆，用于生产可溶性纳米抗体，通过 ELISA 进行鉴定。最终获得了一种高亲和力的抗 EDNRB 纳米抗体，由 119 个氨基酸组成（分子量：12.97 kDa），CDR3 区含有 22 个氨基酸，具有良好的体外亲和力。这些结果提示该纳米抗体可能在人类黑色素瘤的治疗中具有潜在应用价值。
