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 17 000,with the purity of about 95％after purification.BF-42,BF-46 at the concentration of 0.16 μg/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 450 μg/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.
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