人呼吸道合胞病毒纳米抗体的构建及活性  

Construction and activity of respiratory syncytial virus nanobodies

作　　者：王岑嵘 赵慧[2] 卢井才 袁若森 宋月爽 王亚军 王宇迪 姜春来[1] 魏巍 吴山力[4] WANG Cenrong;ZHAO hui;LU Jingcai;YUAN Ruosen;SONG Yueshuang;WANG Yajun;WANG Yudi;JIANG Chunlai;WEI Wei;WU Shanli(College of Life Sciences,Jilin University,Changchun 130012,Jilin Province,China;不详)

机构地区：[1]吉林大学生命科学学院,吉林长春130012 [2]中国食品药品检定研究院呼吸道病毒疫苗室,北京102629 [3]长春百克生物科技股份公司,吉林长春130012 [4]吉林大学基础医学院,吉林长春130021

出　　处：《中国生物制品学杂志》2024年第4期447-452,共6页Chinese Journal of Biologicals

基　　金：吉林省科技发展计划(201603038YY)。

摘　　要：目的 通过免疫羊驼和噬菌体建库筛选出人呼吸道合胞病毒(respiratory syncytial virus,RSV)纳米抗体(nanobodies,Nbs),并评价其结合活性和中和活性。方法 使用RSV重组F蛋白免疫2只羊驼,构建噬菌体文库,筛选得到纳米抗体的骨架区(framework region,FR)和抗原互补决定区(complementarity determining region,CDR)序列,将序列重组构建出3株RSV纳米抗体,使用Ni-NTA亲和层析纯化后,ELISA法检测纳米抗体的结合活性,RSV A2株评价纳米抗体的中和活性。结果 3株纳米抗体BF-42、BF-46、Fh-24相对分子质量约为17 000,纯化后抗体纯度约为95%;BF-42、BF-46浓度为0.16μg/mL,Fh-24浓度为32 ng/mL时仍有结合活性,三者结合能力存在剂量依赖性;3株纳米抗体浓度为450μg/mL时,均可保护50%Hep2细胞免受病毒感染,出现50%致细胞病变效应(cytopathic effect,CPE),能够有效中和病毒。结论 构建的3株纳米抗体具有较高的结合活性和一定的中和能力,为RSV纳米抗体的应用奠定了基础。Objective To screen nanobodies(Nbs)against respiratory syncytial virus(RSV)by immunizing llama and phage library construction,and to evaluate their binding activity and neutralizing ability.Methods Two llamas were immunized with RSV recombinant F protein to construct phage library,and the FR and CDR sequences of Nbs were screened.Three strains of RSV Nbs were constructed by sequence recombination.After purification by Ni⁃NTA affinity chroma⁃tography,the binding activity of the Nbs was detected by ELISA,and the neutralizing activity was evaluated by using RSV A2 strain.Results The three Nbs BF⁃42,BF⁃46 and Fh⁃24 had the relative molecular mass of about 17000,with the purity of about 95%after purification.BF⁃42,BF⁃46 at the concentration of 0.16µg/mL and Fh⁃24 at the concentration of 32 ng/mL still exhibited binding activity,and the binding ability of the three was in a dose⁃dependent manner.At a concentration of 450µg/mL,all the three Nbs protected 50% of Hep2 cells from virus infection,and 50%cytopathic effect(CPE)appeared,which effectively neutralized viruses.Conclusion The three Nbs constructed in this study have high binding activity and a certain neutralizing ability,which lays a foundation of the application of RSV Nbs.

关 键 词：呼吸道合胞病毒 纳米抗体 结合活性 中和活性 

分 类 号：Q816[生物学—生物工程]