| 张玉瑶,高艳春,郝珮羽,等.靶向幽门螺杆菌毒力因子HtrA鲨鱼纳米抗体的开发（英文）[J].中国海洋药物,2024,43(6):11-21. |
| 靶向幽门螺杆菌毒力因子HtrA鲨鱼纳米抗体的开发（英文） |
| Development of Shark VNARs Targeting Helicobacter pylori Virulence Factor HtrA |
| 投稿时间：2023-04-11 修订日期：2023-12-18 |
| DOI：10.13400/j.cnki.cjmd.2024.06.010 |
| 中文关键词: 幽门螺杆菌 HtrA 鲨鱼纳米抗体 噬菌体展示技术 |
| English Keywords:Helicobacter pylori HtrA VNAR Phage display technology |
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| 中文摘要: |
| 目的 构建鲨鱼纳米抗体噬菌体展示文库，从中筛选出针对幽门螺杆菌关键毒力因子HtrA的鲨鱼纳米抗体。方法 用重组的HtrA蛋白免疫条纹斑竹鲨，从其外周血白细胞(PBLs)和脾脏组织中克隆鲨鱼纳米抗体（VNAR）的编码序列，构建鲨鱼纳米抗体噬菌体展示文库，并从中筛选出抗HtrA的鲨鱼纳米抗体。利用大肠杆菌表达系统对鲨鱼纳米抗体进行重组表达并纯化，并用ELISA检测了鲨鱼纳米抗体的亲和力和稳定性。结果 成功构建了针对HtrA的纳米抗体噬菌体展示文库，筛选出了两个鲨鱼纳米抗体(G11和1A5)，分别在大肠杆菌BL21(DE3)细胞中表达，纯化后进行进一步鉴定。评价了鲨鱼纳米抗体的亲和力和稳定性，G11和1A5的EC50值分别为21.2 nM和27.3 nM，并且在低pH溶液中表现出较高的稳定性。构建了1A5的双价鲨鱼纳米抗体，明显提高了抗原结合活性。 |
| English Summary: |
| Objective To construct an immune VNAR phage display library, and develop potent VNARs targeting HtrA which is a critical virulence factor of H. pylori from the library. Methods A whitespotted bamboo shark was immunized with recombinant HtrA protein, and the coding sequence of VNARs was cloned from its peripheral blood leukocytes (PBLs) and spleen tissues. VNAR phage display library was constructed, and anti-HtrA VNARs were selected from the library. The recombinant, expression and purification of VNARs were carried out by using Escherichia coli expression system. ELISA was used to detect the affinity and stability of VNARs. Results VNAR phage display library targeting HtrA was constructed, and two VNARs (G11 and 1A5) were selected, expressed in E. coli BL21(DE3) cells, and purified for further characterization. The affinity and stability of VNARs were assessed. The affinity values of G11 and 1A5 were 21.2 nM and 27.3 nM respectively, and they showed high stability in low pH solutions. A bivalent VNAR Bi-1A5 was constructed and show a significant increase in antigen binding affinity. |
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