Screening And Identification Of Avian Influenza Virus NP And HA1 Nanobodies

Avian influenza(AI)is an acute respiratory infectious disease caused by Avian influenza virus(AIV).According to the antigenicity of AIV surface glycoproteins Hemagglutinin(HA)and Neuraminidase(NA),AIV can be divided into 18 HA subtypes(HA1-HA18)and11 NA subtypes(NA1-NA11).In recent years,human infection of H5N6 AIV occurred frequently,which threatened human health.The development of new detection reagents is more conducive to the monitoring of this virus.Nucleoprotein(NP)is the structural protein of AIV,and HA1 protein is located at the“head”of HA protein.Both proteins are immunogenic.They are the main target proteins for the development of antibodies.Traditional antibody requires technical requirements and has unstable biological properties,while Nanobody(Nb)can be prepared quickly and has stability.In this study,we used phage display technology to obtain Nb against NP protein and HA1 protein to conduct a study on the biological activity of Nb.In order to provide a new idea for the prevention and control of H5N6 AI with Nb.Based on the eukaryotic expression of H5N6 AIV-NP and HA1 proteins,the following experiments were carried out.(1)Construction of phage library:Alpaca was immunized with NP and HA1 proteins,and the Ig G antibody titer was determined by indirect ELISA;the variable domain of the heavy chain of heavy chain(VHH)gene was obtained by nested PCR.The recombinant p Comb-VHH was constructed and the phage display library was established.(2)Screening and expression of Nb:with NP and HA1 proteins as target proteins,the phage display was used to panning the library,and the enrichment was monitored by titer test,the positive phage was identified by phage-ELISA and PCR to obtain the target sequence.(3)Expression and purification of Nb:the pc DNA3.1-Fc-Nb recombinant plasmid was constructed,and Nb was obtained by expression and purification.(4)The biological activity of Nb was identified by hemagglutination inhibition test,indirect ELISA,Western blotting and indirect immunofluorescence assay.The results showed that:(1)The titer of Ig G antibody in serum was 1:128,000.The positive rate of phage display library was 100%and the capacity of phage display library was 1.5×10~9cfu/m L.(2)After three rounds of screening,4 strains of HA1-Nb and 3 strains of NP-Nb were obtained.(3)3 strains of Nb were successfully expressed,named HA1-Nb1-HA1-Nb3 and NP-Nb1-NP-Nb3.(4)The hemagglutination inhibition titer of HA1-Nb1 was 1:2~8.HA1-Nb1 could specifically bind to H5N6 AIV-HA1 protein.NP-Nb1had affinity and recognized NP proteins of different subtypes of influenza viruses broadly.Moreover,NP-Nb1 had binding activity with H1N1,H3N2 and H9N2 influenza viruses.The diversity of alpaca derived phage display library was established,and HA1-Nb1 and NP-Nb1 with reaction activity were screened by phage display technology,which laid a foundation for the new idea of H5N6 AIV prevention.