中国肿瘤生物治疗杂志2024，Vol.31Issue(12)：1211-1217,7.DOI:10.3872/j.issn.1007-385x.2024.12.006
人乳头瘤病毒16型纳米抗体的筛选及鉴定
Screening and identification of nanobody against human papillomavirus 16
摘要
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.关键词
人乳头瘤病毒16型/噬菌体展示技术/纳米抗体/亲和力Key words
human papillomavirus 16(HPV16)/phage display technology/nanobody/affinity分类
农业科技引用本文复制引用
王若瑜,白崇智,仲启明,范瑞文,牛林茹,韩鹏程..人乳头瘤病毒16型纳米抗体的筛选及鉴定[J].中国肿瘤生物治疗杂志,2024,31(12):1211-1217,7.基金项目
山西省科学技术厅重点研发计划项目(No.202202130501015) （No.202202130501015）
山西省科学技术厅基础研究计划项目(No.202303021212353) （No.202303021212353）
山西省中医药管理局科研课题(No.2023ZYYA2002) （No.2023ZYYA2002）
山西省卫生健康委员会科研课题(No.2022107) （No.2022107）
