| 王小月,任雯,刘林,等.靶向TfR1鲨鱼纳米抗体的筛选与鉴定[J].中国海洋药物,2025,44(5):35-42. |
| 靶向TfR1鲨鱼纳米抗体的筛选与鉴定 |
| Screening and characterization of shark VNARs targeting TfR1 |
| 投稿时间：2024-03-25 修订日期：2024-04-25 |
| DOI：10.13400/j.cnki.cjmd.2025.05.011 |
| 中文关键词: 鲨鱼纳米抗体 TfR1 血脑屏障 |
| English Keywords:Shark VNAR TfR1 Blood-brain barrier |
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| 中文摘要: |
| 目的 开发靶向转铁蛋白受体1(TfR1)且具有跨血脑屏障转运功能的鲨鱼纳米抗体。方法 提取未经靶抗原免疫的条纹斑竹鲨外周血单个核细胞总RNA,通过PCR技术扩增获得鲨鱼抗体的重链可变区(VNAR)基因,将VNAR构建至噬菌体展示载体中,完成鲨鱼纳米抗体天然文库的构建。扩增天然噬菌体展示文库,采用抗原固相淘选策略,通过三轮“结合-洗脱-扩增”的淘选流程完成噬菌体展示文库的富集,通过噬菌体ELISA鉴定阳性克隆,并进行抗体序列比对以确定靶向TfR1的鲨鱼纳米抗体序列。构建鲨鱼纳米抗体重组表达载体,利用大肠杆菌原核表达系统进行鲨鱼纳米抗体的诱导表达ELISA检测鲨鱼纳米抗体与TfR1重组蛋白的结合能力,竞争性ELISA检测鲨鱼纳米抗体与Tf竞争受体结合表位的情况,免疫荧光技术检测鲨鱼纳米抗体与脑微血管内皮细胞bEnd.3的结合能力,血脑屏障体外实验模型检测鲨鱼纳米抗体的跨血脑屏障转运能力。结果 本研究成功构建了库容量为2.35×109 cfu的鲨鱼纳米抗体天然文库,并具备良好的多样性。经淘选获得两株靶向TfR1的鲨鱼纳米抗体,均具有亲和活性,其中D1具有跨血脑屏障转运功能。结论 本研究成功筛选出靶向TfR1且具有跨血脑屏障转运功能的鲨鱼纳米抗体,为中枢神经系统疾病治疗提供了新的跨血脑屏障药物递送载体。 |
| English Summary: |
| Objective The aim of this study was to develop of shark VNARs targeting transferrin receptor 1 with transport function across the blood-brain barrier. Methods Peripheral blood mononuclear cells from striped sharks were used to extract total RNA, and the heavy chain variable region (VNAR) gene of shark antibody was obtained by PCR amplification. Subsequently, the VNAR was intergrated into phage display vector to construct a na?ve library of shark VNARs. The na?ve phage display library was amplified, the antigen solid phase panning strategy was used to enrich the phage display library through three rounds of "binding - elution - amplification" panning process. Positive clones were identified through phage ELISA, and the antibody sequences were analyzed to determine shark VNARs sequences targeting TfR1. Recombinant expression vectors for shark VNARs were then constructed, and their expression was induced using the prokaryotic expression system of Escherichia coli. The binding ability of shark VNARs to TfR1 recombinant protein was detected via ELISA. Additionally, the binding epitopes of shark VNARs to Tf competitive receptors were detected using competitive ELISA, while the binding ability of shark VNARs to brain microvascular endothelial cells bEnd.3 was detected by immunofluorescence technology. The ability of shark nanoantibodies to transport across the blood-brain barrier was tested in vitro. Results In this study, a na?ve shark nanobody library with a library capacity of 2.35×109 cfu was successfully constructed, demonstrating good diversity. Two shark VNARs targeting TfR1 were obtained and exhibited affinity activity. Notably, D1 demonstrated trans-blood-brain barrier transport function. Conclusion This study successfully screened shark VNARs targeting TfR1 with trans-blood-brain barrier transport function, providing a novel trans-blood-brain barrier drug delivery vector for the treatment of central nervous system diseases. |
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