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A high affinity nanobody against endothelin receptor type B: a new approach to the treatment of melanoma.

抗内皮素 B 型受体的高亲和力纳米抗体: 治疗黑色素瘤的新方法。

  • 影响因子:2.06
  • DOI:10.1007/s11033-020-05313-w
  • 作者列表:"Ji L","Dong C","Fan R","Qi S
  • 发表时间:2020-02-20
Abstract

: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 VHH 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 × 108/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.

摘要

: 本研究的目的是产生一种在黑色素瘤中高表达的内皮素 B 受体 (EDNRB) 特异性单域抗体 (纳米抗体)。用培养的人黑色素瘤细胞作为抗原免疫羊驼。在羊驼血清中验证抗体生成后,从羊驼淋巴细胞中提取总 RNA,通过两步 PCR 扩增目标 VHH 片段,克隆到 pCANTAB5E phacemid 载体中, 并用于转化大肠杆菌 TG1 细胞,获得噬菌体展示纳米抗体文库,经淘选富集。结果表明,噬菌体展示抗人黑色素瘤 A375 纳米抗体文库构建成功,大小为 1.2 × 108/ml,插入率为 80%。筛选后,用 8 个抗 EDNRB 纳米抗体阳性克隆感染大肠杆菌 HB2151 生产可溶性纳米抗体,用 ELISA 法进行鉴定。最后,我们获得了一种高亲和力的抗 EDNRB 纳米抗体,它由 119 个氨基酸 (分子量: 12.97 kDa) 组成,CDR3 中有 22 个氨基酸,在体外具有良好的亲和力。结果表明,纳米抗体可能用于人类黑色素瘤的治疗。

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