[摘 要] 目的：构建人乳头瘤病毒16型（HPV16） L1蛋白纳米抗体初级文库，通过筛选鉴定获得一株 HPV16 L1特异性纳米 抗体。方法：以HPV 16 L1蛋白为抗原对羊驼进行免疫，采用噬菌体展示技术构建初级抗体文库。经3轮淘选，采用ELISA法鉴定 阳性克隆，将阳性反应最强克隆的VHH序列进行真核表达。经亲和纯化、凝胶过滤层析纯化、SDS?PAGE和WB法鉴定，获得目的 纳米抗体；采用表面等离子共振（SPR）技术检测纳米抗体与HPV 16 L1蛋白之间的亲和力，CCK-8法检测纳米抗体对人永生化角质 细胞HaCat的毒性，荧光素酶报告基因实验检测纳米抗体对HPV 16假病毒的中和活性。结果：初级文库库容为1.304 × 1010 ，丰度 为6.5 × 109 个/mL，ELISA法鉴定获得36个阳性克隆。表达、纯化获得蛋白单体与二聚体，经鉴定为目的纳米抗体（命名为Nb）。Nb 与HPV 16 L1蛋白结合的亲和力为35.41 nmol/L。Nb实验组HaCat细胞增殖活力与空白组没有显著差异（P > 0.05）。与阴性组比 较，0.1和1 μmol/L Nb均能抑制假病毒感染293FT细胞（均P < 0.01）。结论：成功获得一株纯度较好、亲和力较高，对上皮细胞没有 明显毒性作用、有效抑制HPV 16假病毒感染293FT细胞的纳米抗体Nb，为防治HPV 16感染提供了有效的候选抗体类药物。

[Abstract] Objective: To construct a primary nanobody library for human papillomavirus 16 (HPV16) L1 protein and obtain a nanobody specific to HPV16 L1 through selection and identification. Methods: HPV16 L1 protein was used as antigen to immunize alpaca, and a primary antibody library was constructed using phage display technology. After three rounds of screening, positive clones were identified by ELISA. The VHH sequence of the strongest positive clone was used for eukaryotic expression. The target nanobody was obtained after affinity purification, gel filtration chromatography, SDS PAGE and WB identification. The affinity between the nanobody and HPV16 L1 protein was evaluated using surface plasmon resonance (SPR) technology. The cytotoxicity of the nanobody was detected using CCK-8 assay. The neutralizing activity of nanobody against HPV16 pseudovirus was detected using a luciferase reporter gene assay. Results: The primary library was constructed with a capacity of 1.304 × 1010 and an abundance of 6.5 × 109 clones / mL. ELISA identified 36 positive clones. Protein monomer and dimers were expressed and purified, and the target nanobody (designated as "Nb") was successfully identified. The binding affinity of Nb to HPV16 L1 protein was 35.41 nmol/L. There was no significant difference in HaCat cell proliferation activity between Nb group and blank group (P > 0.05). Compared to the negative group, both 0.1 and 1 μmol/L Nb inhibited pseudovirus infection in 293FT cells (all P < 0.01). Conclusion: This study successfully obtained a nanobody with high purity and strong affinity that exhibited no cytotoxicity to epithelial cells and effectively inhibited HPV16 pseudovirus infection in 293FT cells. The nanobody provides a promising candidate antibody-based drug for the prevention and treatment of HPV 16 infection.

山西省科学技术厅重点研发计划项目（No. 202202130501015）；山西省科学技术厅基础研究计划项目（No. 202303021212353）；山西省 中医药管理局科研课题（No. 2023ZYYA2002）；山西省卫生健康委员会科研课题（No. 2022107）