Construction And Screening Of An Influenza Virus Specific Shark Nanobodies Phage Library

Influenza A virus is prone to mutation and has caused worldwide pandemics for many times.At present,there are few drugs for influenza virus,and the limited antiviral drugs and their extensive use have resulted in drug-resistant virus strains.As a new anti-influenza drug,antibody has a good antiviral effect.However,due to the large molecular weight,poor tissue penetration and high production cost,traditional antibody has limited its effectiveness and wide application as a drug.Therefore,finding natural small molecule antibody with good stability and strong affinity has become a new way for the development of antibody drugs.Based on the existing shark nanobody platform in the laboratory,this project used H1N1,H3N2,H5N6 and H7N9 influenza viruses to cross-immunize 3 sharks to obtain high neutralizing titer of shark serum to influenza virus.The maximum semi-inhibitory titers of neutralizing H1N1,H3N2,H5N6 and H7N9 viruses were 2560,2560,640 and 160 times,respectively.We isolated PBMCs from multiple blood samples with high neutralizing titers,extracted RNA and reversed transcription into c DNA,and further utilized phage display technology to successfully construct a phage library with a library capacity of 2×10~8and 98.3%diversity.Broad-spectrum neutralizing antibodies usually recognize conserved epitopes of different antigens,so we used single antigen panning and cross-pannning strategies to obtain broad-spectrum shark nanobodies against influenza virus.Through multiple rounds of solid phase panning,we obtained 80 shark nanobodies sequences using four inactivated virus antigens of H1N1,H3N2,H5N6 and H7N9,and further constructed these sequences into prokaryotic expression vector,and transformed them into EScherichia coli Arctic expression strain.Soluble shark nanobodies were induced by isopropyl thiogalactoside(IPTG).SDS-PAGE was used to detect the different expression level of antibodies in the broken supernatant.Meanwhile,ELISA was used to detect the binding activity of nanobodies in the supernatant of bacteria extractions.79 antibodies binding to the screened antigen were preliminarily screened out.Among them,12 antibodies had good binding to the four inactivated virus antigens of H1N1,H3N2,H5N6 and H7N9.The results showed that the EC50 of 7 shark nanobodies against 7 influenza viruses were all lower than 10μg/m L.The EC50 of the four shark nanoibodies against the seven influenza viruses were all lower than 5μg/m L.Further neutralization experiments showed that the IC50 of two shark nanobodies strains against six influenza viruses were all lower than 2μg/m L.In this project,several influenza virus shark nanobodies with broad spectrum binding activity and neutralization activity were screened and obtained,which laid a certain foundation for further research and development of influenza virus prevention and treatment of antibody drugs.