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

作　　者：王若瑜 白崇智[1,2] 仲启明 范瑞文[2] 牛林茹[3] 韩鹏程 WANG Ruoyu;BAI Chongzhi;ZHONG Qiming;FAN Ruiwen;NIU Linru;HAN Pengcheng(Central Laboratory,Shanxi Provincial Institute of Traditional Chinese Medicine,Taiyuan 030000,Shanxi,China;Alpaca Bioengineering Laboratory,Shanxi Agricultural University,Taigu 030801,Shanxi,China;Junyan Biosciences Co.,Ltd.,Taiyuan 030000,Shanxi,China;School of Medicine,Zhongda Hospital,Southeast University,Nanjin 210000,Jiangsu,China)

机构地区：[1]山西省中医药研究院中心实验室,山西太原030000 [2]山西农业大学羊驼生物工程实验室,山西太谷030801 [3]君研生物科技(山西)有限公司,山西太原030000 [4]东南大学附属中大医院医学院,江苏南京210000

出　　处：《中国肿瘤生物治疗杂志》2024年第12期1211-1217,共7页Chinese Journal of Cancer Biotherapy

基　　金：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)。

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1.304×10^(10),丰度为6.5×10^(9)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1μmol/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。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 1.304×10^(10) and an abundance of 6.5×10^(9) 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 1μmol/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 

分 类 号：S852[农业科学—基础兽医学]