Isolation And Immunological Characteristics Preliminary Analysis Of H5N1 AIV-Specific Nanobodies

Avian influenza(AI)is caused by avian influenza virus(AIV)infection.It’s an acute,highly contagious disease which mainly harms poultry and has brought serious economic losses to the poultry industry in China.AIV can also spread across species,posing a serious threat to human health.H5N1 AIV belongs to highly pathogenic avian influenza virus(HPAIV)that poses a significant threat to humans and poultry,causing systemic transmission of the virus.Currently,vaccination and antiviral drugs are effective prevention and control measures for H5N1 AIV.However,ordinary inactivated vaccines and drugs cannot effectively prevent and control mutated virus strains because of the constant antigenic drift of the virus.Therefore,the research and development of broad-spectrum specific innovative drugs against H5N1 AIV infection and the efficient detection methods for pathogens are particularly important.At present,antibody has become an important material basis for the research and development of human disease diagnosis technologies and drugs.However,the high production cost and the difficult genetic modification of traditional antibodies,their application and research in the prevention and control of animal diseases are difficult.Compared to traditional antibodies,nanobody shows the advantages of unique structure,small molecular weight,ease of genetic modification,mass expression in vitro,low production cost,and simple delivery routes,making it excellent in animal disease diagnosis and control.Based on this,this study used phage display technology to purify H5N1inactivated virus particles as the target antigen to screen and identify specific nanobodies against H5N1 AIV.Then we analyzed the binding effect of nanobodies to H5N1 AIV protein,and the competitive effect of nanobodies against positive chicken serum,the recognized viral antigen regions,and the neutralizing activity in vitro.It provides a new strategy for the development of H5N1 AIV detection methods and antiviral drugs.The main results of this study are described as follows:1.Screened 17 specific nanobodies against H5N1 AIVThe inactivated H5N1 AIV vaccine was used as an immunogen to immunize a4-year-old camel.After 5 times of immunization,indirect ELISA results showed that the titer of H5N1 AIV-specific antibody in serum is 1:128,000.And then isolate lymphocytes from camel peripheral blood,extract total RNA,reverse transcript,amplify VHH gene and a phage nanobody library was constructed by basic molecular biology techniques with a capacity of 2.2×10~8.Then selected 17 specific nanobodies against H5N1 AIV successfully,named Nb4,-5,-6,-17,-20,-25,-33,-36,-40,-43,-53,-54,-56,-58,-70,-78 and-96.2.4 nanobodies against H5N1 AIV recognize the NP protein and 2 nanobodies recognize the NA protein of the virusUsing the expression platform of nanobody-HRP fusion protein,the 17 nanobodies screened above were constructed onto pCMV-N1-HRP vector.Express and prepare the fusion protein of 17 nanobodies and HRP against H5N1 AIV successfully by the eukaryotic expression system of HEK 293T cells.Subsequently,using the fusion protein as a primary antibody,identified 6 nanobodies that bind to H5N1 AIV by IFA,named Nb5,-20,-43,-53,-54,-70.Then expressed the NP protein of H5N1 AIV using prokaryotic and eukaryotic expression systems.ELISA and IFA identified there are 4 nanobodies recognized NP protein using nanobody and HRP fusion proteins as the primary antibody,namely,Nb20,-43,-53,and-70.In addition,construct eukaryotic expression vectors for HA and NA,expressing by HEK 293T cells.IFA identified 2 nanobodies that recognize NA protein using nanobodies as primary antibodies,namely,Nb5 and Nb54.3.Preliminary analysis of immunological characteristics for 6 nanobodiesNP protein can be used as effective targets for antigen detection as the high conservation.In this study,the NP protein expressed in prokaryotic expression was used as a target antigen,identified the competitive effects of Nb20,-43,-53,and-70 against H5N1AIV-positive chicken serum by competitive ELISA.The results showed that the blocking rates of the positive serum against Nb20,-43,-53,and-70 were 72.41%,65.30%,59.24%,and 46.81%,respectively.Subsequently,we truncated the NP protein and expressed them.It was identified that Nb20,-43,-53,and-70 all recognize the 1-62 amino acid region of NP using Western blot.Then,a preliminary analysis was conducted on the neutralizing activity against H5N1 AIV of Nb5,-20,-43,-53,-54,and-70 in vitro.The results showed that Nb5 and Nb54 had significant neutralizing activity against H5N1 AIV.By the way,Nb5 and Nb54 both recognize the NA protein of the virus.Then,the Fc fragment of human Ig G was fusion expressed with Nb54.The fusion protein Nb54-Fc also had a significant neutralization effect in a dose-dependent manner.In summary,the study successfully screened 17 specific nanobodies against H5N1AIV,and identified 6 nanobodies can specifically bind to H5N1 AIV.4 nanobodies can specifically bind to NP protein and can be blocked by H5N1 AIV positive chicken serum.The other 2 nanobodies specifically recognize NA protein and have the activity of neutralizing viral infections in vitro.In brief,this study provides material support for the development of H5N1 AIV diagnostic technology and preventive drugs.